Induction of tumor immunity by variants of folate binding protein

ABSTRACT

The present invention is directed to variants of antigens comprising folate binding protein epitopes as a composition associated with providing immunity against a tumor in an individual. The variant is effective in inducing cytotoxic T-lymphocytes but preferably not to the extent that they become sensitive to silencing by elimination, such as by apoptosis, or by anergy, as in unresponsiveness.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 12,422,600filed Apr. 13, 2009, which issued as U.S. Pat. No. 8,258,261, which is adivisional of application Ser. No. 10/094,097, filed Mar. 8, 2002, whichissued as U.S. Pat. No. 7,547,759, which claims priority to, and thebenefit of, U.S. Application No. 60/274,676, filed on Mar. 9, 2001,which is hereby incorporated herein by reference in its entirety.

The government owns rights in the present invention pursuant to UnitedStates Army grant number DAMD 17-94-J-4313.

FIELD OF THE INVENTION

The present invention is directed to the fields of cancer andimmunology. Specifically, the present invention is directed tocompositions and methods for tumor vaccines directed to tumor antigensand is directed to specific epitopes on these antigens that arerecognized by cytotoxic T-lymphocytes (CTL). More specifically, thepresent invention regards compositions and methods for variants offolate binding protein (FBP).

BACKGROUND OF THE INVENTION

Tumor reactive T-cells have been reported to mediate therapeuticresponses against human cancers (Rosenberg et al., 1988). In certaininstances, in human immunotherapy trials with tumor infiltratinglymphocytes (TIL) or tumor vaccines, these responses correlated eitherwith in vitro cytotoxicity levels against autologous tumors (Aebersoldet al., 1991) or with expression of certain HLA-A, B, C gene products(Marincola et al., 1992). Recent studies (Ioannides et al., 1992) haveproposed that in addition to virally encoded and mutated oncogenes,overexpressed self-proteins may elicit some degree of tumor-reactivecytotoxic T-lymphocytes (CTLs) in patients with various malignancies(Ioannides et al., 1992; Ioannides et al., 1993; Brichard et al., 1993;Jerome et al., 1991). Autologous tumor reactive CTLs can be generatedfrom lymphocytes infiltrating ovarian malignant ascites (Ioannides etal., 1991), and overexpressed proteins, such as HER-2, may be targetsfor CTL recognition (Ioannides et al., 1992).

T-cells play an important role in tumor regression in most murine tumormodels. Tumor infiltrating lymphocytes (TIL) that recognize uniquecancer antigens can be isolated from many murine tumors. The adoptivetransfer of these TIL in addition to interleukin-2 can mediate theregression of established lung and liver metastases (Rosenberg et al.,1986). In addition, the secretion of IFN-γ by injected TIL significantlycorrelates with in vivo regression of murine tumors suggestingactivation of T-cells by the tumor antigens (Barth et al., 1991). Theknown ability of TIL to mediate the regression of metastatic cancer in35 to 40% of melanoma patients when adoptively transferred into patientswith metastatic melanoma attests to the clinical importance of theantigens recognized (Rosenberg et al., 1988; Rosenberg, 1992).

Strong evidence that an immune response to cancer exists in humans isprovided by the existence of tumor reactive lymphocytes within melanomadeposits. These lymphocytes, when isolated, are capable of recognizingspecific tumor antigens on autologous and allogeneic melanomas in an MHCrestricted fashion. (Itoh et al., 1986; Muul et al., 1987; Topalian etal., 1989; Darrow et al., 1989; Hom et al., 1991; Kawakami et al., 1992;Hom et al., 1993; O'Neil et al., 1993). TIL from patients withmetastatic melanoma recognize shared antigens includingmelanocyte-melanoma lineage specific tissue antigens in vitro (Kawakamiet al., 1993; Anichini et al. 1993). Anti-melanoma T-cells appear to beenriched in TIL, probably as a consequence of clonal expansion andaccumulation at the tumor site in vivo (Sensi et al., 1993). Thetransduction of T-cells with a variety of genes, such as cytokines, hasbeen demonstrated. T-cells have been shown to express foreign geneproducts. (Blaese, 1993; Hwu et al., 1993; Culver et al., 1991) The factthat individuals mount cellular and humoral responses against tumorassociated antigens suggests that identification and characterization ofadditional tumor antigens is important for immunotherapy of patientswith cancer.

T-cell receptors on CD8⁺ T-cells recognize a complex consisting of anantigenic peptide (9-10 amino acids for HLA-A2), β2 microglobulin andclass I major histocompatibility complex (MHC) heavy chain (HLA-A, B, C,in humans). Peptides generated by digestion of endogenously synthesizedproteins are transported into the endoplastic reticulum, bound to classI MHC heavy chain and β2 microglobulin, and finally expressed in thecell surface in the groove of the class I MHC molecule.

Information on epitopes of self-proteins recognized in the context ofMHC Class I molecules remain limited, despite a few attempts to identifyepitopes capable of in vitro priming and Ag-specific expansion of humanCTLs. For example, peptide epitopes have been proposed which are likelycandidates for binding on particular MHC Class I Ag (Falk et al., 1991),and some studies have attempted to define peptide epitopes which bindMHC Class I antigens.

Synthetic peptides have been shown to be a useful tool for T-cellepitope mapping. However in vivo and in vitro priming of specific CTLshas encountered difficulties (Alexander et al., 1991; Schild et al.,1991; Carbone et al., 1988). It is generally considered that in vitroCTL priming cannot necessarily be achieved with peptide alone, and infact, a high antigen density is thought to be required for peptidepriming (Alexander et al., 1991). Even in the limited instances whenspecific priming was achieved, APC or stimulators were also required athigh densities (Alexander et al., 1991).

Short synthetic peptides have been used either as target antigens forepitope mapping or for induction of in vitro primary and secondary CTLresponses to viral and parasitic Ags (Bednarek et al., 1991; Gammon etal., 1992; Schmidt et al., 1992; Kos and Müllbacher, 1992; Hill et al.,1992). Unfortunately, these studies failed to show the ability ofproto-oncogene peptide analogs to stimulate in vitro human CTLs to lysetumors endogenously expressing these antigens.

Identification of tumor antigens (Ag) and of specific epitopes on theseAg recognized by cytotoxic T-lymphocytes enables the development oftumor vaccines (for review of tumor antigens, see Rosenberg (2000),incorporated by reference herein). Tumor Ag are weak or partial agonistsfor activation of low-avidity (low-affinity) CTL. Attempts to activateCTL by increasing the affinity of peptide for MHC (by modifications inthe anchor residues) has produced mixed successes even with powerful APC(dendritic cells, DC) and added B7 costimulation. Some of the resultingcross-reactive CTL recognized tumors with lower affinity than CTLinduced by wild type Ag.

The limited ability of anchor-fixed immunogens to induce and expandhigh-affinity CTL raises the need for alternative approaches for CTLinduction. One approach to this question is to design immunogens whichactivate “high-affinity” CTL from the existent pool of responders. Inhuman tumor immunlogy, this approach has been successful in someinstances. However, high-affinity CTL are expected to be more sensitiveto silencing by elimination (e.g apoptosis) or by anergy(unresponsiveness or diminished reactivity to a specific antigen).

These processes occur as a consequence of recurrent stimulations with Ag(tumor Ag) and are amplified by a number of cytokines. The generalmechanism of activation induced cell death (AICD) is that repeatedstimulations with an Ag in the presence of cytokines such as IL-2activates cell death pathways. This is because stimulation with Ag andIL-2 transduces a signal which is too strong to induce proliferation andinstead leads to premature senescence. An alternative death pathway,passive cell death (PCD) occurs when cytokines involved in survival(IL-2, IL-4, IL-7, etc.) are withdrawn. Since tumor Ag are self-Ag, thecorresponding responding cells should be even more sensitive to deletionthan CTL responding to foreign Ag, because the body's defense mechanismsare programmed to avoid autoimmunity. There is little known as to howthe survival of responders to tumor Ag can be induced, and how they canbe protected from AICD or PCD.

Preclinical and clinical trials are underway for the utilization oftumor-specific peptide epitopes for melanoma (Rivoltini et al., 1999;Parkhurst et al., 1998; Kawakami et al., 1998; Lustgarten et al., 1997;Zeng et al., 1997; Reynolds et al., 1998; Nestle et al., 1998;Chakraborty et al., 1998; Rosenberg et al., 1998); breast cancer, suchas with MUC1 (Gendler et al., 1998; Xing et al., 1989; Xing et al.,1990; Jerome et al., 1993; Apostolopoulos et al., 1994; Ding et al.,1993; Zhang et al., 1996; Acres et al., 1993; Henderson et al., 1998;Henderson et al., 1996; Samuel et al., 1998; Gong et al., 1997;Apostolopoulos et al., 1995; Pietersz et al., 1998; Lofthouse et al.,1997; Rowse et al., 1998; Gong et al., 1998; Acres et al., 1999;Apostolopoulos et al., 1998; Lees et al., 1999; Xing et al., 1995;Goydos et al., 1996; Reddish et al., 1998; Karanikas et al., 1997), p53(DeLeo, 1998; McCarty et al., 1998; Hurpin et al., 1998; Gabrilovich etal., 1996), and Her-2/neu (Disis and Cheever, 1998; loannides et al.,1993; Fisk et al., 1995; Peoples et al., 1995; Kawashima et al., 1999;Disi et al., 1996); and colon cancer (Kantor et al., 1992; Kantor etal., 1992; Tsang et al., 1995; Hodge et al., 1997; Conry et al., 1998;Kass et al., 1999; Zaremba et al., 1997; Nukaya et al., 1999).

Recently, peptides of folate binding protein (FBP) were recognized bytumor-associated lymphocytes (Peoples et al., 1998; Peoples et al.,1999; Kim et al., 1999). FBP is a membrane-associated glycoproteinoriginally found as a mAb-defined Ag in placenta and trophoblastic cellsbut rarely in other normal tissues (Retrig et al., 1985; Elwood, 1989;Weitman et al., 1992; Garin-Chesa et al., 1993). Of interest, thisprotein has been found in greater than 90% of ovarian and endometrialcarcinomas; in 20-50% of breast, colorectal, lung, and renal cellcarcinomas; and in multiple other tumor types. When present in canceroustissue, the level of expression is usually greater than 20-fold normaltissue expression and has been reported to be as high as 80-90-fold inovarian carcinomas (Li et al., 1996).

U.S. Pat. No. 5,846,538 is directed to immune reactivity to peptides ofHER-2/neu protein for treatment of malignancies.

Folate binding protein provides an ideal target for and satisfies along-felt need in the art for compositions and methods of utilizing thecompositions directed to tumor immunity.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide as a composition ofmatter an antigen comprising a folate binding protein epitope of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7 and SEQ ID NO:8.

It is another object of the present invention to provide a compositioncomprising an antigen which includes a folate binding protein epitope ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, or a combination thereof in apharmaceutically acceptable excipient.

It is another object of the present invention to provide a method forstimulating cytotoxic T-lymphocytes, comprising the step of contactingthe cytotoxic T-lymphocytes with an amount of an antigen comprising afolate binding protein epitope selected from the group consisting of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, and a combination thereof, wherein theamount is effective to stimulate the cytotoxic T-lymphocytes. In aspecific embodiment of the present invention, the cytotoxicT-lymphocytes are located within a human. In another specificembodiment, the method further comprises the step of administering tothe human an antigen comprising a folate binding protein epitopeselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,and a combination thereof. In another specific embodiment of the presentinvention, the epitope is formulated for administration parenterally,topically, or as an inhalant, aerosol or spray.

It is an additional object of the present invention to provide a methodof generating an immune response, comprising the step of administeringto a human a pharmaceutical composition comprising an immunologicallyeffective amount of a composition comprising an antigen comprising afolate binding epitope of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or acombination thereof.

It is another object of the present invention to provide a method ofinducing immunity against a tumor in an individual, comprising the stepsof administering to the individual an antigen comprising a folatebinding protein epitope of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or acombination thereof; and administering to the individual a cancervaccine. In a specific embodiment of the present invention, the anantigen comprising a folate binding protein epitope is administeredprior to the administration of the cancer vaccine. In a specificembodiment of the present invention, an antigen comprising a folatebinding protein epitope is administered subsequent to the administrationof the cancer vaccine. In another specific embodiment of the presentinvention, the antigen comprising a folate binding protein epitope isadministered both prior to and subsequent to the administration of thecancer vaccine. In a further specific embodiment, the cancer vaccinecomprises a polypeptide selected from the group consisting of SEQ IDNO:268 (E39) and SEQ ID NO:269 (E41).

It is another object of the present invention to provide a method ofinducing memory cytotoxic T-lymphocytes in an individual comprising thestep of administering an antigen comprising a folate binding epitope ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, or a combination thereof. In a specificembodiment, the individual is substantially susceptible to recurrence ofcancer.

It is another object of the present invention to provide a method ofproviding immunity against a tumor comprising the step of administeringan antigen comprising a folate binding epitope vaccine of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, or a combination thereof.

It is another object of the present invention to provide a method oftreating an individual for cancer comprising the steps of administeringto the individual a first cancer vaccine; and administering to theindividual a second cancer vaccine comprising a peptide selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or acombination thereof. In a specific embodiment, the first cancer vaccineadministration step precedes the second cancer vaccine administrationstep. In another specific embodiment, the first cancer vaccineadministration step is subsequent to the second cancer vaccineadministration step.

It is an additional object of the present invention to provide apharmaceutical composition comprising an antigen comprising a folatebinding protein epitope selected from the group consisting of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, or a combination thereof in a pharmaceuticallyacceptable excipient.

It is another object of the present invention to provide a method oftreating a proliferative cell disorder in a human, comprisingadministering to the human a therapeutically effective amount of apharmaceutical composition comprising an antigen comprising a folatebinding protein epitope selected from the group consisting of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, or a combination thereof in a pharmaceuticallyacceptable excipient. In a specific embodiment, the proliferative celldisorder is cancer. In an additional specific embodiment, the cancer isbreast cancer, ovarian cancer, endometrial cancer, colorectal cancer,lung cancer, renal cancer, melanoma, kidney cancer, prostate cancer,brain cancer, sarcomas, or a combination thereof.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 demonstrates HLA-A2 stabilization by FBP epitope E39 variants.

FIG. 2A illustrates IFN-γ induction in peripheral blood mononuclearcells (PBMC) with multiple stimulations with J65 or E39.

FIG. 2B illustrates CTL activity in PBMC with multiple stimulations withJ65 or E39.

FIG. 3 illustrates specific interleukin 2 (IL-2) induction in PBMCs bypriming with E39 variants.

FIG. 4 illustrates expansion of PBMCs stimulated with FBP peptide E39and its variants.

FIG. 5 demonstrates expansion of PBMC stimulated with variants of theFBP peptide E39.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more.

The term “antigen” as used herein is defined as an entity which elicitsan immune system response. The term herein may be abbreviated to “Ag.”

The term “cancer” as used herein is defined as a tissue of uncontrolledgrowth or proliferation of cells, such as a tumor. In a specificembodiment, the cancer is an epithelial cancer. In specific embodiments,the cancer is breast cancer, ovarian cancer, endometrial cancer,colorectal cancer, lung cancer, renal cancer, melanoma, kidney cancer,prostate cancer, brain cancer, sarcomas, or a combination thereof. Inspecific embodiments, such cancers in mammals are caused by chromosomalabnormalities, degenerative growth and/or developmental disorders,mitogenic agents, ultraviolet radiation (uv), viral infections,inappropriate tissue expression of a gene, alterations in expression ofa gene, carcinogenic agents, or a combination thereof. The term melanomaincludes, but is not limited to, melanomas, metastatic melanomas,melanomas derived from either melanocytes or melanocyte related nevuscells, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma insitu, superficial spreading melanoma, nodular melanoma, lentigo malignamelanoma, acral lentiginous melanoma, invasive melanoma or familialatypical mole and melanoma (FAM-M) syndrome. The aforementioned cancerscan be treated by methods described in the present application.

The term “epitope” as used herein is defined as a short peptide derivedfrom a protein antigen which binds to an MHC molecule and is recognizedby a particular T cell.

The term “folate binding protein variant” as used herein is defined as afolate binding protein and peptides thereof which are preferablyrecognized by helper T cells or cytotoxic T cells and may be naturallyderived, synthetically produced, genetically engineered, or a functionalequivalent thereof, e.g where one or more amino acids may be replaced byother amino acid(s) or non-amino acid(s) which do not substantiallyaffect function. In specific embodiments, the peptides are epitopeswhich contain alterations, modifications, or changes in comparison toSEQ ID NO:268 (E39) or SEQ ID NO:269 (E41). In further specificembodiments, the variants are of SEQ ID NO:1 through SEQ ID NO:8.

The term “immune response” as used herein refers to a cellular immuneresponse, including eliciting stimulation of T lymphocytes, macrophages,and/or natural killer cells.

The term “immunity” as used herein is defined as the ability to provideresistance to a tumor resulting from exposure to an antigen that is afolate binding protein epitope, such as the folate binding proteinvariants described herein.

The term “vaccine” as used herein is defined as a composition forgenerating immunity to a cancer. In specific embodiments, the cancervaccine is a wild-type epitope of folate binding protein, such as E39(FBP amino acid residues 191-199) (SEQ ID NO:268) or E41 (FBP amino acidresidues 245-253) (SEQ ID NO:269). In other specific embodiments, thecancer vaccine comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, or acombination thereof. In a preferred embodiment, administration of thevaccine alternates the signaling through the T cell receptor, therebyreducing the possibility of apoptosis.

The term “variant” as used herein is defined as a modified or alteredform of a wildtype sequence, such as the folate binding protein E39epitope (SEQ ID NO:268). The variant may contain replacement of at leastone amino acid residue or may contain an altered side chain for at leastone amino acid residue.

II. The Present Invention A. Specific Embodiments

The present invention is directed to folate binding protein tumor Agmodified to attenuate the signaling through T cell receptors, comparedwith a wild-type folate binding protein tumor Ag, particularly forreducing the possibility of apoptosis that results following repeatedexposure to strong antigens. Thus, variants of folate binding proteinepitopes such as E39 (SEQ ID NO:268) and E41 (SEQ ID NO:269), which are“strong” antigens, are modified to act as a “weak” antigen. Thus, thepresent invention utilizes compositions and methods to attenuatesignaling through the T cell receptors.

The invention works as (1) prestimulation prevaccine, to be administeredbefore the tumor Ag; (2) as post vaccine to be given after the tumor Ag;and/or (3) in certain individuals will work as a priming vaccine. Thesituations (1) and (2) are more related to a protective role for SEQ IDNO:6 (J65) and its analogs for tumor reactive CTL. The situation (3) canbe encountered in certain individuals where mutations in thehistocompatibility Ag binding pocket may transform an attenuator into astrong immunogen.

The invention allows protection before and after vaccination of eitherprecursors (stand-in) or activated effectors. In specific embodiments,administration of the variants of folate binding protein providetargeted induction of memory CTL.

The variants described herein, in a particular embodiment SEQ ID NO:6,are intended to attenuate the signaling at recurrent stimulation, thusinducing protection of CTL precursors as of activated T-cells fromapoptosis, thereby enabling the immune response to expand, and, inpreferred embodiments, have important implications in induction ofmemory CTL.

It is well known that the two major arms of the immune system are: (1)cell-mediated immunity with immune T cells; and (2) humoral immunitywith antibodies. Further, the immune system normally functions torecognize and destroy any foreign or aberrant cells in the body. SinceFBP is expressed by some normal cells, tolerance and/or anergy isexpected.

Development of molecular therapies for cancer have historically focusedon specific recognition of Ags by cellular immune effectors. The presentinvention discloses novel strategies aimed at identification of peptidetargets for CTLs, and generation of T-cell immunity against specificepitopes (for a review of T-cell specific immunity, see, e.g, Ioannideset al., 1992; Houbiers et al., 1993).

To achieve this, the present invention provides novel naturally- andsynthetically-derived peptides which bind human leucocyte antigen— (HLA)class I heavy chains. Appropriate criteria for epitope selection invitro have been defined, and synthetic peptides based on immunogenicepitopes of FBP have also been produced.

Although the dominant anchors for peptide binding to HLA-A2 are Leu (P2)and Val (P9), a number of residues with similar charge and side chains,such as Ile and/or Met, were identified in CTL epitopes from viralproteins (Falk et al., 1991; Bednarek et al., 1991).

B. General Embodiments

1. CTL Epitopes

CTL epitopes reported to date are mainly derived from foreign (viral)proteins with little or no homology to self-proteins. With respect toCTL responses to self-proteins, it is expected that T-cells expressingTCR with high affinity for self-peptide-MHC class I complexes areeliminated in the thymus during development. Self-peptides eluted fromHLA-A2.1 molecules of various cell lines show residues at P3-P5 andP7-P8 which are different from the sequences of viral epitopesrecognized by human CTLs. Since these residues are likely to contact andinteract with TCR, they may reflect peptides for which autologousT-cells are already tolerant/anergic.

For T-cell recognizing self-epitopes to be eliminated or anergized, aprecondition exists that the peptide-MHC complex is stable enough toengage a sufficient number of TCRs, or at least more stable than otherHLA-A2 peptide complexes, where one peptide can be easily displaced byother peptides. Consequently, this would suggest that for self-proteinswith extension to FBP, the ones that can bind TCR with high affinityduring development will be less likely to be recognized later whenexpressed on a tumor other target, than peptides that bind HLA-A2 withlow affinity, which under appropriate conditions (e.g, high proteinconcentration) may occupy a higher number of HLA-A2 molecules. Forlow-affinity peptides, modification of the anchors resulting instabilization of peptide —HLA-A2 interaction by replacing weak withdominant anchor residues (e.g, (P9) M

V, should facilitate the reactivity of CTL with targets expressing suchantigens, because TCR interacts mainly with the sequence P4-P8.

Tumor progression and metastasis are often associated withoverexpression of specific cellular proteins. Epitopes of non-mutatedoverexpressed proteins can be targets of a specific cellular immuneresponse against tumor mediated by T-cells. Moreover, when T-cellepitopes are present, distinction between tumor immunity/autoimmunityand unresponsiveness can be predicated on the protein concentration as alimiting factor of epitope supply.

2. Epitopic Core Sequences

The present invention is also directed to protein or peptidecompositions, free from total cells and other peptides, which comprise apurified protein or peptide which incorporates an epitope that isimmunologically recognized by a CTL.

As used herein, the term “incorporating an epitope(s) that isimmunologically recognized by a CTL” is intended to refer to a peptideor protein antigen which includes a primary, secondary or tertiarystructure similar to an epitope located within a FBP polypeptide. Thelevel of similarity will generally be to such a degree that the samepopulation of CTLs will also bind to, react with, or otherwiserecognize, the cross-reactive peptide or protein antigen.

The identification of CTL-stimulating immunodominant epitopes, and/ortheir functional equivalents, suitable for use in vaccines is arelatively straightforward matter. For example, one may employ themethods of Hopp, as taught in U.S. Pat. No. 4,554,101, incorporatedherein by reference, which teaches the identification and preparation ofepitopes from amino acid sequences on the basis of hydrophilicity. Themethods described in several other papers, and software programs basedthereon, can also be used to identify epitopic core sequences (see, forexample, Jameson and Wolf, 1988; Wolf et al., 1988; U.S. Pat. No.4,554,101). The amino acid sequence of these “epitopic core sequences”may then be readily incorporated into peptides, either through theapplication of peptide synthesis or recombinant technology.

Preferred peptides for use in accordance with the present invention willgenerally be on the order of 8 to 20 amino acids in length, and morepreferably about 8 to about 15 amino acids in length. It is proposedthat shorter antigenic CTL-stimulating peptides will provide advantagesin certain circumstances, for example, in the preparation of vaccines orin immunologic detection assays. Exemplary advantages include the easeof preparation and purification, the relatively low cost and improvedreproducibility of production, and advantageous biodistribution.

It is proposed that particular advantages of the present invention maybe realized through the preparation of synthetic peptides which includemodified and/or extended epitopic/immunogenic core sequences whichresult in a “universal” epitopic peptide directed to FBP sequences.These epitopic core sequences are identified herein in particularaspects as hydrophilic regions of the FBP polypeptide antigen. It isproposed that these regions represent those which are most likely topromote T-cell or B-cell stimulation, and, hence, elicit specificantibody production.

An epitopic core sequence, as used herein, is a relatively short stretchof amino acids that is “complementary” to, and therefore will bind,receptors on CTLs. It will be understood that in the context of thepresent disclosure, the term “complementary” refers to amino acids orpeptides that exhibit an attractive force towards each other.

In general, the size of the polypeptide antigen is not believed to beparticularly crucial, so long as it is at least large enough to carrythe identified core sequence or sequences. The smallest useful coresequence anticipated by the present disclosure would generally be on theorder of about 8 amino acids in length, with sequences on the order of 9or 10 being more preferred. Thus, this size will generally correspond tothe smallest peptide antigens prepared in accordance with the invention.However, the size of the antigen may be larger where desired, so long asit contains a basic epitopic core sequence.

A skilled artisan recognizes that numerous computer programs areavailable for use in predicting antigenic portions of proteins (see e.g,Jameson & Wolf, 1988; Wolf et al., 1988). Computerized peptide sequenceanalysis programs (e.g, DNAStar Software, DNAStar, Inc., Madison, Wis.)may also be useful in designing synthetic peptides in accordance withthe present disclosure.

Syntheses of epitopic sequences, or peptides which include an antigenicepitope within their sequence, are readily achieved using conventionalsynthetic techniques such as the solid phase method (e.g, through theuse of commercially available peptide synthesizer such as an AppliedBiosystems Model 430A Peptide Synthesizer). Peptide antigens synthesizedin this manner may then be aliquoted in predetermined amounts and storedin conventional manners, such as in aqueous solutions or, even morepreferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may bereadily stored in aqueous solutions for fairly long periods of time ifdesired, e.g, up to six months or more, in virtually any aqueoussolution without appreciable degradation or loss of antigenic activity.However, where extended aqueous storage is contemplated it willgenerally be desirable to include agents including buffers such as Trisor phosphate buffers to maintain a pH of about 7.0 to about 7.5.Moreover, it may be desirable to include agents which will inhibitmicrobial growth, such as sodium azide or Merthiolate. For extendedstorage in an aqueous state it will be desirable to store the solutionsat 4□C., or more preferably, frozen. Of course, where the peptides arestored in a lyophilized or powdered state, they may be stored virtuallyindefinitely, e.g, in metered aliquots that may be rehydrated with apredetermined amount of water (preferably distilled) or buffer prior touse.

3. T lymphocytes

T lymphocytes recognize antigen in the form of peptide fragments thatare bound to class I and class II molecules of the majorhistocompatibility complex (MHC) locus. Major Histocompatibility Complex(MHC) is a generic designation meant to encompass the histocompatibilityantigen systems described in different species including the humanleucocyte antigens (HLA). The T-cell receptor for antigen (TCR) is acomplex of at least 8 polypeptide chains. (“Basic and ClinicalImmunology” (1994) Stites, Terr and Parslow(eds) Appleton and Lange,Nenmack Conn.) Two of these chains (the alpha and beta chains) form adisulfide-linked dimer that recognizes antigenic peptides bound to MHCmolecules and therefore is the actual ligand-binding structure withinthe TCR. The TCR alpha and beta chains are similar in many respects toimmunoglobulin proteins. The amino-terminal regions of the alpha andbeta chains are highly polymorphic, so that within the entire T-cellpopulation there are a large number of different TCR alpha/beta dimers,each capable of recognizing or binding a particular combination ofantigenic peptide and MHC.

In general, CD4⁺ T cell populations are considered to function ashelpers/inducers through the release of lymphokines when stimulated by aspecific antigen; however, a subset of CD4⁺ cells can act as cytotoxic Tlymphocytes (CTL). Similarly, CD8⁺ T cells are considered to function bydirectly lysing antigenic targets; however, under a variety ofcircumstances they can secrete lymphokines to provide helper or DTHfunction. Despite the potential of overlapping function, the phenotypicCD4 and CD8 markers are linked to the recognition of peptides bound toclass H or class I MHC antigens. The recognition of antigen in thecontext of class II or class I MHC mandates that CD4⁺ and CD8⁺ T cellsrespond to different antigens or the same antigen presented underdifferent circumstances. The binding of immunogenic peptides to class IIMHC antigens most commonly occurs for antigens ingested by antigenpresenting cells. Therefore, CD4⁺ T cells generally recognize antigensthat have been external to the tumor cells. By contrast, under normalcircumstances, binding of peptides to class I MHC occurs only forproteins present in the cytosol and synthesized by the target itself,proteins in the external environment are excluded. An exception to thisis the binding of exogenous peptides with a precise class I bindingmotif which are present outside the cell in high concentration. Thus,CD4⁺ and CD8⁺ T cells have broadly different functions and tend torecognize different antigens as a reflection of where the antigensnormally reside.

As disclosed within the present invention, the protein product expressedby FBP is recognized by T cells. Such a protein expression product“turns over” within cells, i.e., undergoes a cycle wherein a synthesizedprotein functions and then eventually is degraded and replaced by anewly synthesized molecule. During the protein life cycle, peptidefragments from the protein bind to major histocompatibility complex(MHC) antigens. By display of a peptide bound to MHC antigen on the cellsurface and recognition by host T cells of the combination of peptideplus self MHC antigen, a malignant cell will be immunogenic to T cells.The exquisite specificity of the T cell receptor enables individual Tcells to discriminate between protein fragments which differ by a singleamino acid residue.

During the immune response to a peptide, T cells expressing a T cellreceptor with high affinity binding of the peptide-MHC complex will bindto the peptide-MHC complex and thereby become activated and induced toproliferate. In the first encounter with a peptide, small numbers ofimmune T cells will secrete lymphokines, proliferate and differentiateinto effector and memory T cells. Subsequent encounters with the sameantigen by the memory T cell will lead to a faster and more intenseimmune response.

Intact folate binding protein or peptides thereof which are recognizedby cytotoxic T cells may be used within the present invention. Thepeptides may be naturally derived or produced based upon an identifiedsequence. The peptides for CD8⁺ T cell responses (elicited by peptidespresented by folate binding protein class I MHC molecules) are generallyabout 8-10 amino acids in length. Peptides for CD8⁺ T cell responsesvary according to each individual's class I MHC molecules. Examples ofpeptides suitable within the present invention for CD8⁺ T cell responsesinclude peptides comprising or consisting of SEQ ID NO:1 through SEQ IDNO:8.

It will be evident to those of ordinary skill in the art that otherpeptides may be produced for use within the present invention, both forclass I MHC molecules as well as for class II molecules. A variety oftechniques are well known for isolating or constructing peptides.Suitable peptides are readily identified based upon the disclosureprovided herein. Additional suitable peptides include those which arelonger in length. Such peptides may be extended (e.g, by the addition ofone or more amino acid residues and/or truncated (e.g, by the deletionof one or more amino acid residues from the carboxyl terminus).Alternatively, suitable peptides may be variations on other preferredpeptides disclosed herein. Although this particular peptide variationmay result in a peptide with the same number of total amino acids (suchas nine), a peptide variation on a preferred peptide need not beidentical in length. Variations in amino acid sequence that yieldpeptides having substantially the same desired biological activity arewithin the scope of the present invention.

Immunization of an individual with a FBP peptide (i.e., as a vaccine)can induce continued expansion in the number of T cells necessary fortherapeutic attack against a tumor in which FBP is associated.Typically, about 0.01 μg/kg to about 100 mg/kg body weight will beadministered by the intradermal, subcutaneous or intravenous route. Apreferred dosage is about 1 μg/kg to about 1 mg/kg, with about 5 μg/kgto about 200 μg/kg particularly preferred. It will be evident to thoseskilled in the art that the number and frequency of administrations willbe dependent upon the response of the patient. It may be desirable toadminister the FBP peptide repetitively. It will be evident to thoseskilled in this art that more than one FBP peptide may be administered,either simultaneously or sequentially. For example, a combination ofabout 8-15 peptides may be used for immunization. Preferred peptides forimmunization are those that include all or a portion of at least one FBPamino acid SEQ ID NO:1 through SEQ ID NO:68, or variants thereof. One ormore peptides from other portions of the amino acid sequence shown inSEQ ID NO:1 through SEQ ID NO:68 may be added to one or more of thepreferred peptides.

In addition to the FBP peptide (which functions as an antigen), it maybe desirable to include other components in the vaccine, such as avehicle for antigen delivery and immunostimulatory substances designedto enhance the protein's immunogenicity. Examples of vehicles forantigen delivery include aluminum salts, water-in-oil emulsions,biodegradable oil vehicles, oil-in-water emulsions, biodegradablemicrocapsules, and liposomes. Examples of immunostimulatory substances(adjuvants) include N-acetylmuramyl-L-alanine-D-isoglutamine (MDP),lipopoly-saccharides (LPS), glucan, IL-12, GM-CSF, gamma interferon andIL-15. It will be evident to those skilled in this art that a FBPpeptide may be prepared synthetically or that a portion of the protein(naturally-derived or synthetic) may be used. When a peptide is usedwithout additional sequences, it may be desirable to couple the peptidehapten to a carrier substance, such as keyhole limpet hemocyanin.

The methods and compositions of the present invention are particularlywell-suited for inducing an immune response in a patient who hasdeveloped resistance to conventional cancer treatments or who has a highprobability of developing a recurrence following treatment. A skilledartisan recognizes that cancer cells are able to evade the immune systemor evade an effective immune response because they look like self, theyactively anergize the immune system to any antigens which maypotentially differentiate between self and tumor, and they may create animmunosuppressive environment by secreting immunosuppressive factorsand/or by expressing factors which can induce apoptosis of an offensivetumor antigen-specific killer cell.

A skilled artisan is aware of multiple reviews concerning cancervaccines and the generation of cellular immune responses to antigenictumor peptides (Pietersz et al., 2000; Pardoll, 2000; Rosenberg, 2000;Dalgleish, 2000, each of which are incorporated by reference herein).

A skilled artisan recognizes that the antigen can be produced in largeamounts by recombinant technology, either as soluble molecules ineukaryotic systems or as fusion proteins in bacterial systems. In aspecific embodiment, synthetic peptides are made from the tumor antigen.Furthermore, monoclonal antibodies to the tumor antigens are useful intheir identification and purification.

In a peptide approach to tumor immunotherapy, peptides (such as about8-9mers) are presented by MHC class I molecules, leading to thegeneration of CD8⁺-mediated cellular responses comprising CTLs andcytokine secretion, mostly in the form of IFN-γ and TNF-α.

A skilled artisan recognizes that the dendritic cell is important ingenerating CD8⁺ CTLs following class I presentation. Esche et al. (1999)demonstrated techniques whereby dendritic cells are obtained frompatients, isolated, expanded in vitro, exposed to the peptides andreintroduced into the patient. Others utilize similarly treateddendritic cells for generation of specifically activated T cells invitro before transfer.

A crucial initial step in CD8⁺ T cell generation is the uptake andpresentation of peptides by MHC molecules by antigen-presenting cells.MHC class I proteins consist of three subunits, all of which areimportant for the formation of a stable complex. X-ray crystallographyof MHC class I molecules has demonstrated that interaction of peptideswith the MHC class I groove is determined by the peptide sequence, withdiscrete amino acids interacting with pockets in the MHC groove (whichhave a fixed spacing from each other) and also have specificity foranchoring amino acid side chains. Although there are exceptions, theamino and carboxy termini of the peptides are anchored at either end ofthe groove, often in positions 2 or 3, 5 or 7 (Apostolopoulos et al.,1997a; Apostolopoulos et al., 1997b). The peptides also interact withthe T cell receptor, yet only a small amount of the peptide is exposed(Apostolopoulos et al., 1998).

Given that multiple peptide tumor antigens, such as folate bindingprotein, have been identified in addition to characterization of T cellepitopes, in a specific embodiment of the present invention peptideantigens are generated synthetically for immunization. Theimmunogenicity of small peptides can be improved upon by increasing thepeptide size, by binding to carriers and also by using adjuvants toactivate macrophages and other immune system factors. A skilled artisanis cognizant of recombinant cytokines being used to increaseimmunogenicity of a synthetic peptide (Tao and Levy, 1993) andfurthermore that cytokines can also be directly fused to peptides (Nakaoet al., 1994; Disis et al., 1996; Chen et al., 1994).

In specific embodiments of the present invention, mixtures of separatepeptides are administered as a vaccine. Alternatively, multiple epitopesmay be incorporated into the same molecule by recombinant technologywell known in the art (Mateo et al., 1999; Astori and Krachenbuhl,1996). In another embodiment, a combinatorial peptide library is used toincrease binding peptides by utilizing different amino acids at leastone anchor location.

In another embodiment of the present invention, natural amino acids of apeptide are replaced with unnatural D-amino acids; alternatively, thepeptide residues are assembled in reverse order, which renders thepeptides resistant to proteases (Briand et al., 1997; Herve et al.,1997; Bartnes et al., 1997; Guichard et al., 1996). In anotherembodiment, unnatural modified amino acids are incorporated into apeptide, such as α-aminoisobutyric acid or N-methylserine.

A skilled artisan recognizes that the binding strength of the 8- or9-mer to the MHC complex and the subsequent recognition by the T cellreceptor determines the immunogenicity of CTL peptides. Van Der Burg etal. (1993) determined that the longer the peptide remains bound to theMHC complex, the better the chance it will induce a T cell response. Askilled artisan also recognizes that there are methods for introducingextraneous peptides directly into the cytoplasm of a cell to allowgeneration of class I-restricted cellular immune responses. One exampleincludes microbial toxins, which can carry peptides in their cytoplasmfor delivery because they enter cells by receptor-mediated endocytosisand thereby deposit cellular toxins into the cytoplasm. Specificexamples include shiga toxin (Lee et al., 1998), anthrax toxin (Goletzet al., 1997), diphtheria toxin (Stenmark et al., 1991), Pseudomonasexotoxin (Donnelly et al., 1993), and Bordetella pertussis toxin(Fayolle et al., 1996).

In alternative embodiments, peptides enter cells through membrane fusionand are beneficial for delivering tumor or other peptides into a cellcytoplasm, including Antennapedia (Derossi et al., 1994; Derossi et al.,1996; Schutze-Redelmeier et al., 1996), Tat protein (Kim et al., 1997),and Measles virus fusion peptide (Partidos et al., 1997).

In other embodiments, peptides are introduced into a cytoplasm throughlipopeptides, which comprise both a lipid and a peptide, by directinsertion into the lipophilic cell membrane (BenMohamed et al., 1997;Obert et al., 1998; Deprez et al., 1996; Beekman et al., 1997). Inalternative embodiments, the peptides are delivered in liposomes (forexamples, see Nakanishi et al., 1997; Noguchi et al., 1991; Fukasawa etal., 1998; Guan et al., 1998), whereby the immunogenicity is dependenton the size, charge, lipid composition of the liposome itself, andwhether or not the antigen is present on the surface of the liposome orwithin its interior.

A skilled artisan also recognizes that immune-stimulating complexes(ISCOMs), which comprise Quill A (a mixture of saponins), cholesterol,phospholipid, and proteins, are useful for delivering naturallyhydrophobic antigens or antigens made hydrophobic by the addition ofmyristic or palmitic acid tails (for examples, see Hsu et al., 1996;Sjolander et al., 1997; Villacres-Eriksson, 1995; Tarpey et al., 1996;Rimmelzwaan et al., 1997). ISCOMs facilitate penetration into cells byfusion with their membranes, by endocytosis, or by phagocytosis.

Antigens may also be directed to particular subcellular compartmentsthrough incorporation of sorting signals to the antigen by recombinanttechnology, including Class II LAMP-I (Rowell et al., 1995; Wu et al.,1995), ER targeting peptide (Minev et al., 1994); CLIP (Malcherik etal., 1998), and heat shock proteins (Udono and Srivastava, 1993; Heikeet al., 1996; Zhu et al., 1996; Suzue et al., 1997; Ciupitu et al.,1998).

A skilled artisan recognizes that the present invention providesanti-cancer therapeutic compositions comprising a variety of peptidesdesignated for CD8⁺ T cell responses comprising SEQ ID NO:1 through SEQID NO:8, or a combination thereof. A skilled artisan also recognizesthat the present invention provides anti-cancer therapeutic compositionscomprising a variety of peptides designated for CD8⁺ T cell responsesconsisting essentially of SEQ ID NO:1 through SEQ ID NO:8, or acombination thereof.

A skilled artisan recognizes that references such as Abrams and Schlom(2000) summarize the current views on rational Ag modification. Twotypes of peptides are described: (1) agonistic peptides which upregulateAg-specific responses; (2) antagonistic/partial agonistic peptides whichdownregulate the same responses. However, it is an object of the presentinvention to provide therapy which stimulate Ag-specific immuneresponses while at the same time does not elicit activation induced-celldeath or death by neglect.

A skilled artisan recognizes that sequences that encode folate bindingprotein epitopes for induction of tumor immunity can be obtained fromdatabases such as the National Center for Biotechnology Informations'sGenBank® database or commercially available databases, such as that ofCelera Genomics, Inc. (Rockville, Md.). Examples of folate bindingprotein sequences which may comprise an epitope or which can be alteredto comprise an epitope include the following, denoted by GenBank®Accession numbers: P14207 (SEQ ID NO:9); P15328 (SEQ ID NO:10); P13255(SEQ ID NO:11); NP_(—)000793 (SEQ ID NO:12); AAB05827 (SEQ ID NO:13);AAG36877 (SEQ ID NO:14); 542627 (SEQ ID NO:15); 500112 (SEQ ID NO:16);BFBO (SEQ ID NO:17); 562670 (SEQ ID NO:18); S62669 (SEQ ID NO:19);A55968 (SEQ ID NO:20); A45753 (SEQ ID NO:21); A33417 (SEQ ID NO:22);B40969 (SEQ ID NO:23); A40969 (SEQ ID NO:24); NP_(—)057943 (SEQ IDNO:25); NP_(—)057942 (SEQ ID NO:26); NP_(—)057941 (SEQ ID NO:27);NP_(—)057937 (SEQ ID NO:28); NP_(—)057936 (SEQ ID NO:29); NP_(—)037439(SEQ ID NO:30); NP_(—)032061 (SEQ ID NO:31); NP_(—)032060 (SEQ IDNO:32); NP_(—)000795 (SEQ ID NO:33); NP_(—)000794 (SEQ ID NO:34);AAF66225 (SEQ ID NO:35); BAA37125 (SEQ ID NO:36); PO2752 (SEQ ID NO:37);Q05685 (SEQ ID NO:38); P35846 (SEQ ID NO:39); PO2702 (SEQ ID NO:40);AAD53001 (SEQ ID NO:41); AAD33741 (SEQ ID NO:42); AAD33740 (SEQ IDNO:43); AAD19354 (SEQ ID NO:44); AAD19353 (SEQ ID NO:45); AAC98303 (SEQID NO:46); AAB81938 (SEQ ID NO:47); AAB81937 (SEQ ID NO:48); AAB49703(SEQ ID NO:49); AAB35932 (SEQ ID NO:50); 1011184A (SEQ ID NO:51);0908212A (SEQ ID NO:52); CAA44610 (SEQ ID NO:53); CAA83553 (SEQ IDNO:54); AAA74896 (SEQ ID NO:55); AAA49056 (SEQ ID NO:56); AAA37599 (SEQID NO:57); AAA37598 (SEQ ID NO:58); AAA37597 (SEQ ID NO:59); AAA37594(SEQ ID NO:60); AAA37596 (SEQ ID NO:61); AAA37595 (SEQ ID NO:62);AAA35824 (SEQ ID NO:63); AAA35823 (SEQ ID NO:64); AAA35822 (SEQ IDNO:65); AAA35821 (SEQ ID NO:66); AAA18382 (SEQ ID NO:67); and AAA17370(SEQ ID NO:68).

A skilled artisan also recognizes that epitopes of folate bindingprotein, nucleic acid sequences are encoded by, or altered to encode avariant of, for example, one of the following: U02715 (SEQ ID NO:69);BE518506 (SEQ ID NO:70); BG058247 (SEQ ID NO:71); BG017460 (SEQ IDNO:72); NM_(—)000802 (SEQ ID NO:73); U20391 (SEQ ID NO:74); NM_(—)016731(SEQ ID NO:75); NM_(—)016730 (SEQ ID NO:76); NM_(—)016729 (SEQ IDNO:77); NM_(—)016725 (SEQ ID NO:78); NM_(—)016724 (SEQ ID NO:79);NM_(—)013307 (SEQ ID NO:80); NM_(—)008035 (SEQ ID NO:81); NM_(—)008034(SEQ ID NO:82); BF153292 (SEQ ID NO:83); BF114518 (SEQ ID NO:84);BE940806 (SEQ ID NO:85); BE858996 (SEQ ID NO:86); AF219906 (SEQ IDNO:87); AF219905 (SEQ ID NO:88); AF219904 (SEQ ID NO:89); BE687177 (SEQID NO:90); BE636622 (SEQ ID NO:91); BE627230 (SEQ ID NO:92); BE506561(SEQ ID NO:93); BE505048 (SEQ ID NO:94); BE496754 (SEQ ID NO:95);BB114010 (SEQ ID NO:96); BB109527 (SEQ ID NO:97); BB107219 (SEQ IDNO:98); BE206324 (SEQ ID NO:99); BE448392 (SEQ ID NO:100); BE207596 (SEQID NO:101); BE206635 (SEQ ID NO:102); BE240998 (SEQ ID NO:103); BE228221(SEQ ID NO:104); BE225416 (SEQ ID NO:105); BE225404 (SEQ ID NO:106);BB214040 (SEQ ID NO:107); BE199619 (SEQ ID NO:108); BE199597 (SEQ IDNO:109); BE198610 (SEQ ID NO:110); BE198571 (SEQ ID NO:111); BE188055(SEQ ID NO:112); BE187804 (SEQ ID NO:113); BB032646 (SEQ ID NO:114);BE037278 (SEQ ID NO:115); BE037125 (SEQ ID NO:116); BE037110 (SEQ IDNO:117); BE037009 (SEQ ID NO:118); BE036024 (SEQ ID NO:119); BE035828(SEQ ID NO:120); BE035751 (SEQ ID NO:121); BE019724 (SEQ ID NO:122);AW913291 (SEQ ID NO:123); AW912445 (SEQ ID NO:124); AW823912 (SEQ IDNO:125); AW823418 (SEQ ID NO:126); AB023803 (SEQ ID NO:127); AB022344(SEQ ID NO:128); AW475385 (SEQ ID NO:129); AW323586 (SEQ ID NO:130);AW319308 (SEQ ID NO:131); AW239668 (SEQ ID NO:132); AV253136 (SEQ IDNO:133); AW013716 (SEQ ID NO:134); AW013704 (SEQ ID NO:135); AW013702(SEQ ID NO:136); AW013696 (SEQ ID NO:137); AW013669 (SEQ ID NO:138);AW013647 (SEQ ID NO:139); AW013501 (SEQ ID NO:140); AW013484 (SEQ IDNO:141); AW013428 (SEQ ID NO:142); AW013404 (SEQ ID NO:143); AW013386(SEQ ID NO:144); AW013284 (SEQ ID NO:145); AW013183 (SEQ ID NO:146);AF061256 (SEQ ID NO:147); AI956572 (SEQ ID NO:148); AI882550 (SEQ IDNO:149); AI822932 (SEQ ID NO:150); AI785988 (SEQ ID NO:151); AI744273(SEQ ID NO:152); AI727302 (SEQ ID NO:153); AI725714 (SEQ ID NO:154);AF137375 (SEQ ID NO:155); AF137374 (SEQ ID NO:156); AF137373 (SEQ IDNO:157); AF096320 (SEQ ID NO:158); AF096319 (SEQ ID NO:159); AI663857(SEQ ID NO:160); AI647841 (SEQ ID NO:161); AI646950 (SEQ ID NO:162);AI607910 (SEQ ID NO:163); AI529173 (SEQ ID NO:164); AI509734 (SEQ IDNO:165); AI506267 (SEQ ID NO:166); AI498269 (SEQ ID NO:167); AI000444(SEQ ID NO:168); AA956337 (SEQ ID NO:169); AA955042 (SEQ ID NO:170);AA899838 (SEQ ID NO:171); AA899718 (SEQ ID NO:172); AA858756 (SEQ IDNO:173); AI311561 (SEQ ID NO:174); AI385951 (SEQ ID NO:175); AI352406(SEQ ID NO:176); AF100161 (SEQ ID NO:177); AI326503 (SEQ ID NO:178);AI325517 (SEQ ID NO:179); AI325453 (SEQ ID NO:180); AI325382 (SEQ IDNO:181); AI323700 (SEQ ID NO:182); AI323374 (SEQ ID NO:183); AI313973(SEQ ID NO:184); AI196928 (SEQ ID NO:185); AF091041 (SEQ ID NO:186);AI156212 (SEQ ID NO:187); AI120374 (SEQ ID NO:188); AI119000 (SEQ IDNO:189); AA408670 (SEQ ID NO:190); AA408072 (SEQ ID NO:191); AA407615(SEQ ID NO:192); AA995272 (SEQ ID NO:193); C78593 (SEQ ID NO:194);AA999910 (SEQ ID NO:195); AA991491 (SEQ ID NO:196); X99994 (SEQ IDNO:197); X99993 (SEQ ID NO:198); X99992 (SEQ ID NO:199); X99991 (SEQ IDNO:200); X99990 (SEQ ID NO:201); AA958985 (SEQ ID NO:202); AA873222 (SEQID NO:203); AA930051 (SEQ ID NO:204); AA895334 (SEQ ID NO:205); AA796142(SEQ ID NO:206); AA798223 (SEQ ID NO:207); AA734325 (SEQ ID NO:208);AA690871 (SEQ ID NO:209); AA674988 (SEQ ID NO:210); AA674863 (SEQ IDNO:211); AA674821 (SEQ ID NO:212); AA674744 (SEQ ID NO:213); AA671558(SEQ ID NO:214); AF000381 (SEQ ID NO:215); AF000380 (SEQ ID NO:216);AA637071 (SEQ ID NO:217); AA616314 (SEQ ID NO:218); AA109687 (SEQ IDNO:219); AA608235 (SEQ ID NO:220); AA589050 (SEQ ID NO:221); AA544782(SEQ ID NO:222); AA522095 (SEQ ID NO:223); AA386821 (SEQ ID NO:224);AA386818 (SEQ ID NO:225); AA386495 (SEQ ID NO:226); AA289278 (SEQ IDNO:227); AA286342 (SEQ ID NO:228); AA276302 (SEQ ID NO:229); AA276123(SEQ ID NO:230); AA277280 (SEQ ID NO:231); AA273543 (SEQ ID NO:232);U89949 (SEQ ID NO:233); AA208306 (SEQ ID NO:234); AA208089 (SEQ IDNO:235); AA242285 (SEQ ID NO:236); AA139715 (SEQ ID NO:237); AA139709(SEQ ID NO:238); AA139675 (SEQ ID NO:239); AA139593 (SEQ ID NO:240);AA124010 (SEQ ID NO:241); AA108790 (SEQ ID NO:242); AA108350 (SEQ IDNO:243); AA028831 (SEQ ID NO:244); AA061275 (SEQ ID NO:245); W82933 (SEQID NO: 246); AA015571 (SEQ ID NO:247); W71715 (SEQ ID NO:248); W59165(SEQ ID NO:249); X62753 (SEQ ID NO:250); Z32564 (SEQ ID NO:251); T29279(SEQ ID NO:252); M25317 (SEQ ID NO:253); M86438 (SEQ ID NO:254); J03922(SEQ ID NO:255); M64817 (SEQ ID NO:256); L25338 (SEQ ID NO:257); M97701(SEQ ID NO:258); M97700 (SEQ ID NO:259); M64782 (SEQ ID NO:260); M35069(SEQ ID NO:261); J05013 (SEQ ID NO:262); M28099 (SEQ ID NO:263); J02876(SEQ ID NO:264); U08471 (SEQ ID NO:265); U02714 (SEQ ID NO:266); andU02716 (SEQ ID NO:267).

A skilled artisan also recognizes that the scope of the invention is notlimited to the specific nonapeptides described in SEQ ID NO:1 throughSEQ ID NO:8. The antigens comprising a FBP epitope may be at least about7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or up to about30. It is contemplated that any amino acid may be used for additions orfilling in for the remainder of sequences in addition to the specificvariant sequence provided herein. However, it is preferred that theywill be those that will maintain the underlying sequence of FBP.

III. Rational Vaccine Design

The goal of rational vaccine design is to produce structural analogs ofbiologically active compounds. By creating such analogs, it is possibleto fashion vaccines which are more active or stable than the naturalmolecules, which have different susceptibility to alteration or whichmay affect the function of various other molecules. In one approach, askilled artisan generates a three-dimensional structure for the folatebinding protein variant of the invention or a fragment thereof. Thiscould be accomplished by X-ray crystallography, computer modeling, or bya combination of both approaches. An alternative approach involves therandom replacement of functional groups throughout the folate bindingprotein variant, and the resulting affect on function is determined.

It also is possible to isolate a folate binding protein variant specificantibody, selected by a functional assay, and then solve its crystalstructure. In principle, this approach yields a pharmacore upon whichsubsequent vaccine design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies to afunctional, pharmacologically active antibody. As a mirror image of amirror image, the binding site of anti-idiotype would be expected to bean analog of the original antigen. The anti-idiotype could then be usedto identify and isolate peptides from banks of chemically- orbiologically-produced peptides. Selected peptides would then serve asthe vaccine.

Thus, one may design vaccines which have enhanced and improvedbiological activity, for example, anti-tumor activity, relative to astarting folate binding protein variant of the invention. By virtue ofstandard chemical isolation procedures and other descriptions herein,sufficient amounts of the folate binding protein variants of theinvention can be produced to perform crystallographic studies. Inaddition, knowledge of the chemical characteristics of these compoundspermits computer-employed predictions of structure-functionrelationships.

IV. Immunological Reagents

It is well known in the art that the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitableadjuvants include all acceptable immunostimulatory compounds, such ascytokines, chemokines, cofactors, toxins, plasmodia, syntheticcompositions or LEEs or CEEs encoding such adjuvants.

Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12,γ-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such asthur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A(MPL). RIBI, which contains three components extracted from bacteria,MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2%squalene/Tween 80 emulsion is also contemplated. MHC antigens may evenbe used. Exemplary, often preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

In addition to adjuvants, it may be desirable to coadminister biologicresponse modifiers (BRM), which have been shown to upregulate T cellimmunity or down-regulate suppressor cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA);low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, NJ), cytokinessuch as g-interferon, IL-2, or IL-12 or genes encoding proteins involvedin immune helper functions, such as B-7.

A variety of routes can be used to administer the vaccines including butnot limited to subcutaneous, intramuscular, intradermal, intraepidermal,intravenous and intraperitoneal.

An individual, such as a patient, is injected with vaccine generally asdescribed above. The antigen may be mixed with adjuvant, such asFreund's complete or incomplete adjuvant. Booster administrations withthe same vaccine or DNA encoding the same may occur at approximatelytwo-week intervals.

V. Immunotherapeutic Agents

An immunotherapeutic agent generally relies on the use of immuneeffector cells and molecules to target and destroy cancer cells. Theimmune effector may be, for example, a folate binding protein variantwhich is or is similar to a tumor cell antigen. The variant alone mayserve as an effector of therapy or it may recruit other cells toactually effect cell killing. The variant also may be conjugated to adrug or toxin (e.g, a chemotherapeutic, a radionuclide, a ricin A chain,a cholera toxin, a pertussis toxin, etc.) and serve merely as atargeting agent. Such antibody conjugates are called immunotoxins, andare well known in the art (see U.S. Pat. No. 5,686,072, U.S. Pat. No.5,578,706, U.S. Pat. No. 4,792,447, U.S. Pat. No. 5,045,451, U.S. Pat.No. 4,664,911, and U.S. Pat. No. 5,767,072, each incorporated herein byreference). Alternatively, the effector may be a lymphocyte carrying asurface molecule that interacts, either directly or indirectly, with atumor cell target. Various effector cells include cytotoxic T cells andNK cells.

In one aspect of immunotherapy, the tumor cell must bear some markerthat is amenable to targeting, i.e., is not present on the majority ofother cells. Many tumor markers exist in addition to folate bindingprotein described herein, and any of these may be suitable for targetingin the context of the present invention. Common tumor markers includecarcinoembryonic antigen, prostate specific antigen, urinary tumorassociated antigen, fetal antigen, tyrosinase (p9′7), gp68, TAG-72,HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155.

The disclosures presented herein have significant relevance toimmunotherapy of human diseases and disorders, including cancer. Inusing the immunotherapeutic compositions derived from the antigen of thepresent invention in treatment methods, other standard treatments alsomay be employed, such as radiotherapy or chemotherapy. However, it ispreferred that the immunotherapy be used alone initially as itseffectiveness can be readily assessed. Immunotherapies of cancer canbroadly be classified as adoptive, passive and active, as described inthe following sections, and may be used or produced with the folatebinding protein variant antigen of the present invention.

A. Immune Stimulators

A specific aspect of immunotherapy is to use an immune stimulatingmolecule as an agent, or more preferably in conjunction with anotheragent, such as, for example, a cytokine such as IL-2, IL-4, IL-12,GM-CSF, tumor necrosis factor; interferons alpha, beta, and gamma; F42Kand other cytokine analogs; a chemokine such as, for example, MIP-1,MIP-1beta, MCP-1, RANTES, IL-8; or a growth factor such as, for example,FLT3 ligand.

One particular cytokine contemplated for use in the present invention istumor necrosis factor. Tumor necrosis factor (TNF; Cachectin) is aglycoprotein that kills some kinds of cancer cells, activates cytokineproduction, activates macrophages and endothelial cells, promotes theproduction of collagen and collagenases, is an inflammatory mediator andalso a mediator of septic shock, and promotes catabolism, fever andsleep. Some infectious agents cause tumor regression through thestimulation of TNF production. TNF can be quite toxic when used alone ineffective doses, so that the optimal regimens probably will use it inlower doses in combination with other drugs. Its immunosuppressiveactions are potentiated by gamma-interferon, so that the combinationpotentially is dangerous. A hybrid of TNF and interferon-α also has beenfound to possess anti-cancer activity.

Another cytokine specifically contemplate is interferon alpha.Interferon alpha has been used in treatment of hairy cell leukemia,Kaposi's sarcoma, melanoma, carcinoid, renal cell cancer, ovary cancer,bladder cancer, non-Hodgkin's lymphomas, mycosis fungoides, multiplemyeloma, and chronic granulocytic leukemia.

B. Passive Immunotherapy

A number of different approaches for passive immunotherapy of cancerexist. They may be broadly categorized into the following: injection ofvaccine alone; injection of vaccine coupled to toxins orchemotherapeutic agents; injection of vaccine coupled to radioactiveisotopes; injection of anti-idiotype vaccine; and finally, purging oftumor cells in bone marrow.

It may be favorable to administer more than one vaccine associated withtwo different antigens or even vaccine with multiple antigenspecificity. Treatment protocols also may include administration oflymphokines or other immune enhancers (Bajorin et al. 1988).

C. Active Immunotherapy

In some embodiments of the invention, active immunotherapy may beemployed. In active immunotherapy, a folate binding protein variant(e.g, a peptide or polypeptide), a nucleic acid encoding a folatebinding protein variant, and/or additional vaccine components, such asfor example, a cell expressing the folate binding protein variant (e.g adendritic cell fused with a tumor cell, or an autologous or allogeneictumor cell composition expressing the antigen), an adjuvant, arecombinant protein, an immunomodulator, and the like is administered(Ravindranath and Morton, 1991; Morton and Ravindranath, 1996; Morton etal., 1992; Okamoto et al., 1997; Kugler et al., 2000; Trefzer et al.,2000; Mitchell et al., 1990; Mitchell et al., 1993).

An antigenic peptide, polypeptide or protein, or an autologous orallogenic tumor cell composition or “vaccine” is administered generallywith a distinct bacterial adjuvant (Ravindranath and Morton, 1991;Morton and Ravindranath, 1996; Morton et al., 1992; Mitchell et al.,1990; Mitchell et al., 1993). In melanoma immunotherapy, those patientswho elicit high IgM response often survive better than those who elicitno or low IgM antibodies (Morton et al., 1992). IgM antibodies are oftentransient antibodies and the exception to the rule appears to beanti-ganglioside or anti-carbohydrate antibodies.

D. Adoptive Immunotherapy

In adoptive immunotherapy, the patient's circulating lymphocytes, ortumor infiltrated lymphocytes, are isolated in vitro, activated bylymphokines such as IL-2 or transduced with genes for tumor necrosis,and readministered (Rosenberg et al., 1988; 1989). To achieve this, onewould administer to an animal, or human patient, an immunologicallyeffective amount of activated lymphocytes in combination with anadjuvant-incorporated antigenic peptide composition as described herein.The activated lymphocytes will most preferably be the patient's owncells that were earlier isolated from a blood or tumor sample andactivated (or “expanded”) in vitro. In certain embodiments, thepatient's lymphocytes are cultured or expanded in number or selected foractivity, such as immunoreactivity to the antigen. This form ofimmunotherapy has produced several cases of regression of melanoma andrenal carcinoma.

VI. Vaccines

The present invention contemplates vaccines for use in both active andpassive immunization embodiments. Immunogenic compositions, proposed tobe suitable for use as a vaccine, may be prepared most readily directlyfrom immunogenic CTL-stimulating peptides prepared in a manner disclosedherein. Preferably the antigenic material is extensively dialyzed toremove undesired small molecular weight molecules and/or lyophilized formore ready formulation into a desired vehicle.

The preparation of vaccines which contain peptide sequences as activeingredients is generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;and 4,578,770, all incorporated herein by reference. Typically, suchvaccines are prepared as injectables. Either as liquid solutions orsuspensions: solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified. The active immunogenic ingredient is often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,or adjuvants which enhance the effectiveness of the vaccines.

Vaccines may be conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkalene glycols or triglycerides: such suppositories maybe formed from mixtures containing the active ingredient in the range ofabout 0.5% to about 10%, preferably about 1 to about 2%. Oralformulations include such normally employed excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain about 10to about 95% of active ingredient, preferably about 25 to about 70%.

The peptides of the present invention may be formulated into the vaccineas neutral or salt forms. Pharmaceutically-acceptable salts, include theacid addition salts (formed with the free amino groups of the peptide)and those which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups may also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g, the capacity of the individual's immunesystem to synthesize antibodies, and the degree of protection desired.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges areof the order of several hundred micrograms active ingredient pervaccination. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These arebelieved to include oral application on a solid physiologicallyacceptable base or in a physiologically acceptable dispersion,parenterally, by injection or the like. The dosage of the vaccine willdepend on the route of administration and will vary according to thesize of the host.

Various methods of achieving adjuvant effect for the vaccine includesuse of agents such as aluminum hydroxide or phosphate (alum), commonlyused as about 0.05 to about 0.1% solution in phosphate buffered saline,admixture with synthetic polymers of sugars (Carbopol®) used as an about0.25% solution, aggregation of the protein in the vaccine by heattreatment with temperatures ranging between about 70° to about 101° C.for a 30-second to 2-minute period, respectively. Aggregation byreactivating with pepsin treated (Fab) antibodies to albumin, mixturewith bacterial cells such as C. parvum or endotoxins orlipopolysaccharide components of Gram-negative bacteria, emulsion inphysiologically acceptable oil vehicles such as mannide mono-oleate(Aracel A) or emulsion with a 20% solution of a perfluorocarbon(Fluosol-DA®) used as a block substitute may also be employed.

In many instances, it will be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and preferably one or more, usually at leastabout three vaccinations. The vaccinations will normally be at from twoto twelve week intervals, more usually from three to five weekintervals. Periodic boosters at intervals of 1-5 years, usually threeyears, will be desirable to maintain protective levels of theantibodies. The course of the immunization may be followed by assays forantibodies for the supernatant antigens. The assays may be performed bylabeling with conventional labels, such as radionuclides, enzymes,fluorescents, and the like. These techniques are well known and may befound in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932;4,174,384 and 3,949,064, as illustrative of these types of assays.

For an antigenic composition to be useful as a vaccine, an antigeniccomposition must induce an immune response to the antigen in a cell,tissue or animal (e.g, a human). As used herein, an “antigeniccomposition” may comprise an antigen (e.g, a peptide or polypeptide), anucleic acid encoding an antigen (e.g, an antigen expression vector), ora cell expressing or presenting an antigen. In particular embodiments,the antigenic composition comprises or encodes a folate binding proteinvariant, or an immunologically functional equivalent thereof. In otherembodiments, the antigenic composition is in a mixture that comprises anadditional immunostimulatory agent or nucleic acids encoding such anagent. Immunostimulatory agents include but are not limited to anadditional antigen, an immunomodulator, an antigen presenting cell or anadjuvant. In other embodiments, one or more of the additional agent(s)is covalently bonded to the antigen or an immunostimulatory agent, inany combination. In certain embodiments, the antigenic composition isconjugated to or comprises an HLA anchor motif amino acids.

In certain embodiments, an antigenic composition or immunologicallyfunctional equivalent, may be used as an effective vaccine in inducingan anti-folate binding protein variant humoral and/or cell-mediatedimmune response in an animal. The present invention contemplates one ormore antigenic compositions or vaccines for use in both active andpassive immunization embodiments.

A vaccine of the present invention may vary in its composition ofproteinaceous, nucleic acid and/or cellular components. In anon-limiting example, a nucleic acid encoding an antigen might also beformulated with a proteinaceous adjuvant. Of course, it will beunderstood that various compositions described herein may furthercomprise additional components. For example, one or more vaccinecomponents may be comprised in a lipid or liposome. In anothernon-limiting example, a vaccine may comprise one or more adjuvants. Avaccine of the present invention, and its various components, may beprepared and/or administered by any method disclosed herein or as wouldbe known to one of ordinary skill in the art, in light of the presentdisclosure.

A. Proteinaceous Antigens

It is understood that an antigenic composition of the present inventionmay be made by a method that is well known in the art, including but notlimited to chemical synthesis by solid phase synthesis and purificationaway from the other products of the chemical reactions by HPLC, orproduction by the expression of a nucleic acid sequence (e.g, a DNAsequence) encoding a peptide or polypeptide comprising an antigen of thepresent invention in an in vitro translation system or in a living cell.Preferably the antigenic composition is isolated and extensivelydialyzed to remove one or more undesired small molecular weightmolecules and/or lyophilized for more ready formulation into a desiredvehicle. It is further understood that additional amino acids,mutations, chemical modification and the like, if any, that are made ina vaccine component will preferably not substantially interfere with theantibody recognition of the epitopic sequence.

A peptide or polypeptide corresponding to one or more antigenicdeterminants of the folate binding protein variant of the presentinvention should generally be at least five or six amino acid residuesin length, and may contain up to about 10, about 15, about 20, or more.A peptide sequence may be synthesized by methods known to those ofordinary skill in the art, for example, peptide synthesis usingautomated peptide synthesis machines, such as those available fromApplied Biosystems (Foster City, Calif.).

Longer peptides or polypeptides also may be prepared, e.g, byrecombinant means. In certain embodiments, a nucleic acid encoding anantigenic composition and/or a component described herein may be used,for example, to produce an antigenic composition in vitro or in vivo forthe various compositions and methods of the present invention. Forexample, in certain embodiments, a nucleic acid encoding an antigen iscomprised in, for example, a vector in a recombinant cell. The nucleicacid may be expressed to produce a peptide or polypeptide comprising anantigenic sequence. The peptide or polypeptide may be secreted from thecell, or comprised as part of or within the cell.

B. Genetic Vaccine Antigens

In certain embodiments, an immune response may be promoted bytransfecting or inoculating an animal with a nucleic acid encoding anantigen. One or more cells comprised within a target animal thenexpresses the sequences encoded by the nucleic acid after administrationof the nucleic acid to the animal. Thus, the vaccine may comprise“genetic vaccine” useful for immunization protocols. A vaccine may alsobe in the form, for example, of a nucleic acid (e.g, a cDNA or an RNA)encoding all or part of the peptide or polypeptide sequence of anantigen. Expression in vivo by the nucleic acid may be, for example, bya plasmid type vector, a viral vector, or a viral/plasmid constructvector.

In preferred aspects, the nucleic acid comprises a coding region thatencodes all or part of the sequences disclosed as SEQ ID NO:1 throughSEQ ID NO:9, or an immunologically functional equivalent thereof. Ofcourse, the nucleic acid may comprise and/or encode additionalsequences, including but not limited to those comprising one or moreimmunomodulators or adjuvants. The nucleotide and protein, polypeptideand peptide encoding sequences for various genes have been previouslydisclosed, and may be found at computerized databases known to those ofordinary skill in the art. One such database is the National Center forBiotechnology Information's Genbank® and GenPept databases. The codingregions for these known genes may be amplified, combined with thenucleic acid sequences encoding the folate binding protein variantdisclosed herein (e.g, ligated) and/or expressed using the techniquesdisclosed herein or by any technique that would be know to those ofordinary skill in the art (e.g, Sambrook et al., 1987). Though a nucleicacid may be expressed in an in vitro expression system, in preferredembodiments the nucleic acid comprises a vector for in vivo replicationand/or expression.

C. Cellular Vaccine Antigens

In another embodiment, a cell expressing the antigen may comprise thevaccine. The cell may be isolated from a culture, tissue, organ ororganism and administered to an animal as a cellular vaccine. Thus, thepresent invention contemplates a “cellular vaccine.” The cell may betransfected with a nucleic acid encoding an antigen to enhance itsexpression of the antigen. Of course, the cell may also express one ormore additional vaccine components, such as immunomodulators oradjuvants. A vaccine may comprise all or part of the cell.

D. Immunologically Functional Equivalents

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thefollowing codon table:

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg RAGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU ThreonineThr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode the peptideswithout appreciable loss of their biological utility or activity. Aminoacid substitutions may be based on the relative similarity of the aminoacid side-chain substituents, for example, their hydrophobicity,hydrophilicity, charge, size, and the like. Exemplary substitutionswhich take various of the foregoing characteristics into considerationare well known to those of skill in the art.

Numerous scientific publications have also been devoted to theprediction of secondary structure, and to the identification of anepitope, from analyses of an amino acid sequence (Chou and Fasman,1974a,b; 1978a,b, 1979). Any of these may be used, if desired, tosupplement the teachings of U.S. Pat. No. 4,554,101.

Moreover, computer programs are currently available to assist withpredicting an antigenic portion and an epitopic core region of one ormore proteins, polypeptides or peptides. Examples include those programsbased upon the Jameson-Wolf analysis (Jameson & Wolf, 1988; Wolf et al.,1988), the program PepPlot.®. (Brutlag et al., 1990; Weinberger et al.,1985), and other new programs for protein tertiary structure prediction(Fetrow & Bryant, 1993). Another commercially available software programcapable of carrying out such analyses is MacVector® (IBI, New Haven,Conn.).

As modifications and changes may be made in the structure of anantigenic composition (e.g, a folate binding protein variant) of thepresent invention, and still obtain molecules having like or otherwisedesirable characteristics, such immunologically functional equivalentsare also encompassed within the present invention.

For example, certain amino acids may be substituted for other aminoacids in a peptide, polypeptide or protein structure without appreciableloss of interactive binding capacity with structures such as, forexample, antigen-binding regions of antibodies, binding sites onsubstrate molecules or receptors, DNA binding sites, or such like. Sinceit is the interactive capacity and nature of a peptide, polypeptide orprotein that defines its biological (e.g, immunological) functionalactivity, certain amino acid sequence substitutions can be made in aamino acid sequence (or, of course, its underlying DNA coding sequence)and nevertheless obtain a peptide or polypeptide with like (agonistic)properties. It is thus contemplated by the inventors that variouschanges may be made in the sequence of an antigenic composition such as,for example a folate binding protein variant peptide or polypeptide, orunderlying DNA, without appreciable loss of biological utility oractivity.

Accordingly, antigenic composition, particularly an immunologicallyfunctional equivalent of the sequences disclosed herein, may encompassan amino molecule sequence comprising at least one of the 20 commonamino acids in naturally synthesized proteins, or at least one modifiedor unnatural amino acid, including but not limited to those shown onTable 2 below.

TABLE 2 Modified, Unnatural or Rare Amino Acids Abbr. Amino Acid Abbr.Amino Acid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine Baad3-Aminoadipic acid Hyl Hydroxylysine Bala β-alanine, b-Amino- AhylAllo-Hydroxylysine propionic acid Abu 2-Aminobutyric acid 3Hyp3-Hydroxyproline 4Abu 4-Aminobutyric acid, 4Hyp 4-Hydroxyprolinepiperidinic acid Acp 6-Aminocaproic acid Ide Isodesmosine Ahe2-Aminoheptanoic acid Aile Allo-Isoleucine Aib 2-Aminoisobutyric acidMeGly N-Methylglycine, sarcosine Baib 3-Aminoisobutyric acid MeIleN-Methylisoleucine Apm 2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu2,4-Diaminobutyric acid MeVal N-Methylvaline Des Desmosine Nva NorvalineDpm 2,2′-Diaminopimelic acid Nle Norleucine Dpr 2,3-Diaminopropionicacid Orn Ornithine EtGly N-Ethylglycine

In terms of immunologically functional equivalent, it is well understoodby the skilled artisan that, inherent in the definition is the conceptthat there is a limit to the number of changes that may be made within adefined portion of the molecule and still result in a molecule with anacceptable level of equivalent immunological activity. Animmunologically functional equivalent peptide or polypeptide are thusdefined herein as those peptide(s) or polypeptide(s) in which certain,not most or all, of the amino acid(s) may be substituted.

In particular, where a shorter length peptide is concerned, it iscontemplated that fewer amino acid substitutions should be made withinthe given peptide. A longer polypeptide may have an intermediate numberof changes. The full-length protein will have the most tolerance for alarger number of changes. Of course, a plurality of distinctpolypeptides/peptides with different substitutions may easily be madeand used in accordance with the invention.

It also is well understood that where certain residues are shown to beparticularly important to the immunological or structural properties ofa protein or peptide, e.g, residues in binding regions or active sites,such residues may not generally be exchanged. This is an importantconsideration in the present invention, where changes in the folatebinding protein variant antigenic site should be carefully consideredand subsequently tested to ensure maintenance of immunological function(e.g, antigenicity), where maintenance of immunological function isdesired. In this manner, functional equivalents are defined herein asthose peptides or polypeptides which maintain a substantial amount oftheir native immunological activity.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. An analysisof the size, shape and type of the amino acid side-chain substituentsreveals that arginine, lysine and histidine are all positively chargedresidues; that alanine, glycine and serine are all a similar size; andthat phenylalanine, tryptophan and tyrosine all have a generally similarshape. Careful selection of a particular amino acid substitution for apeptide, as opposed to a protein, must be considered given thedifferences in size between peptides and proteins.

In further embodiments, major antigenic determinants of a peptide orpolypeptide may be identified by an empirical approach in which portionsof a nucleic acid encoding a peptide or polypeptide are expressed in arecombinant host, and the resulting peptide(s) or polypeptide(s) testedfor their ability to elicit an immune response. For example, PCR™ can beused to prepare a range of peptides or polypeptides lacking successivelylonger fragments of the C-terminus of the amino acid sequence. Theimmunoactivity of each of these peptides or polypeptides is determinedto identify those fragments or domains that are immunodominant. Furtherstudies in which only a small number of amino acids are removed at eachiteration then allows the location of the antigenic determinant(s) ofthe peptide or polypeptide to be more precisely determined.

Another method for determining a major antigenic determinant of apeptide or polypeptide is the SPOTs™ system (Genosys Biotechnologies,Inc., The Woodlands, Tex.). In this method, overlapping peptides aresynthesized on a cellulose membrane, which following synthesis anddeprotection, is screened using a polyclonal or monoclonal antibody. Anantigenic determinant of the peptides or polypeptides which areinitially identified can be further localized by performing subsequentsyntheses of smaller peptides with larger overlaps, and by eventuallyreplacing individual amino acids at each position along theimmunoreactive sequence.

Once one or more such analyses are completed, an antigenic composition,such as for example a peptide or a polypeptide is prepared that containat least the essential features of one or more antigenic determinants.An antigenic composition is then employed in the generation of antiseraagainst the composition, and preferably the antigenic determinant(s).

While discussion has focused on functionally equivalent polypeptidesarising from amino acid changes, it will be appreciated that thesechanges may be effected by alteration of the encoding DNA; taking intoconsideration also that the genetic code is degenerate and that two ormore codons may code for the same amino acid. Nucleic acids encodingthese antigenic compositions also can be constructed and inserted intoone or more expression vectors by standard methods (Sambrook et al.,1987), for example, using PCR™ cloning methodology.

In addition to the peptidyl compounds described herein, the inventorsalso contemplate that other sterically similar compounds may beformulated to mimic the key portions of the peptide or polypeptidestructure or to interact specifically with, for example, an antibody.Such compounds, which may be termed peptidomimetics, may be used in thesame manner as a peptide or polypeptide of the invention and hence arealso immunologically functional equivalents.

Certain mimetics that mimic elements of protein secondary structure aredescribed in Johnson et al. (1993). The underlying rationale behind theuse of peptide mimetics is that the peptide backbone of proteins existschiefly to orientate amino acid side chains in such a way as tofacilitate molecular interactions, such as those of antibody andantigen. A peptide mimetic is thus designed to permit molecularinteractions similar to the natural molecule.

E. Antigen Mutagenesis

In particular embodiments, an antigenic composition is mutated forpurposes such as, for example, enhancing its immunogenicity or producingor identifying an immunologically functional equivalent sequence.Methods of mutagenesis are well known to those of skill in the art(Sambrook et al., 1987).

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

In a preferred embodiment, site directed mutagenesis is used.Site-specific mutagenesis is a technique useful in the preparation of anantigenic composition (e.g, a folate binding protein variant-comprisingpeptide or polypeptide, or immunologically functional equivalentprotein, polypeptide or peptide), through specific mutagenesis of theunderlying DNA. In general, the technique of site-specific mutagenesisis well known in the art. The technique further provides a ready abilityto prepare and test sequence variants, incorporating one or more of theforegoing considerations, by introducing one or more nucleotide sequencechanges into the DNA. Site-specific mutagenesis allows the production ofa mutant through the use of specific oligonucleotide sequence(s) whichencode the DNA sequence of the desired mutation, as well as a sufficientnumber of adjacent nucleotides, to provide a primer sequence ofsufficient size and sequence complexity to form a stable duplex on bothsides of the position being mutated. Typically, a primer of about 17 toabout 75 nucleotides in length is preferred, with about 10 to about 25or more residues on both sides of the position being altered, whileprimers of about 17 to about 25 nucleotides in length being morepreferred, with about 5 to 10 residues on both sides of the positionbeing altered.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. As will be appreciated by one of ordinary skill in theart, the technique typically employs a bacteriophage vector that existsin both a single stranded and double stranded form. Typical vectorsuseful in site-directed mutagenesis include vectors such as the M13phage. These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

This mutagenic primer is then annealed with the single-stranded DNApreparation, and subjected to DNA polymerizing enzymes such as, forexample, E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected that include recombinant vectors bearing the mutatedsequence arrangement.

Alternatively, a pair of primers may be annealed to two separate strandsof a double stranded vector to simultaneously synthesize bothcorresponding complementary strands with the desired mutation(s) in aPCR™ reaction. A genetic selection scheme to enrich for clonesincorporating the mutagenic oligonucleotide has been devised (Kunkel etal., 1987). Alternatively, the use of PCR™ with commercially availablethermostable enzymes such as Taq polymerase may be used to incorporate amutagenic oligonucleotide primer into an amplified DNA fragment that canthen be cloned into an appropriate cloning or expression vector (Tomicet al., 1990; Upender et al., 1995). A PCR™ employing a thermostableligase in addition to a thermostable polymerase also may be used toincorporate a phosphorylated mutagenic oligonucleotide into an amplifiedDNA fragment that may then be cloned into an appropriate cloning orexpression vector (Michael 1994).

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

Additionally, one particularly useful mutagenesis technique is alaninescanning mutagenesis in which a number of residues are substitutedindividually with the amino acid alanine so that the effects of losingside-chain interactions can be determined, while minimizing the risk oflarge-scale perturbations in protein conformation (Cunningham et al.,1989).

F. Vectors

In order to effect replication, expression or mutagenesis of a nucleicacid, the nucleic acid may be delivered (“transfected”) into a cell. Thetranfection of cells may be used, in certain embodiments, torecombinately produce one or more vaccine components for subsequentpurification and preparation into a pharmaceutical vaccine. In otherembodiments, the nucleic acid may be comprised as a genetic vaccine thatis administered to an animal. In other embodiments, the nucleic acid istransfected into a cell and the cell administered to an animal as acellular vaccine component. The nucleic acid may consist only of nakedrecombinant DNA, or may comprise, for example, additional materials toprotect the nucleic acid and/or aid its targeting to specific celltypes.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g, YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques (see, for example, Maniatis et al., 1988 and Ausubel et al.,1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for a RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell.

The nucleic acid encoding the antigenic composition or other vaccinecomponent may be stably integrated into the genome of the cell, or maybe stably maintained in the cell as a separate, episomal segment of DNA.Such nucleic acid segments or “episomes” encode sequences sufficient topermit maintenance and replication independent of or in synchronizationwith the host cell cycle. Vectors and expression vectors may containnucleic acid sequences that serve other functions as well and aredescribed infra. How the expression construct is delivered to a cell andwhere in the cell the nucleic acid remains is dependent on the type ofexpression construct employed.

1. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as, for example, thepromoter for the mammalian terminal deoxynucleotidyl transferase geneand the promoter for the SV40 late genes, a discrete element overlyingthe start site itself helps to fix the place of initiation. Additionalpromoter elements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30-110 bp upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include theβ-lactamase (penicillinase), lactose and tryptophan (trp) promotersystems. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202 and 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al. 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

Additionally any promoter/enhancer combination (as per, for example, theEukaryotic Promoter Data Base EPDB) could also be used to driveexpression. Use of a T3, T7 or SP6 cytoplasmic expression system isanother possible embodiment. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct.

Table 3 lists non-limiting examples of elements/promoters that may beemployed, in the context of the present invention, to regulate theexpression of a RNA. Table 4 provides non-limiting examples of inducibleelements, which are regions of a nucleic acid sequence that can beactivated in response to a specific stimulus.

TABLE 3 Promoter and/or Enhancer Promoter/Enhancer ReferencesImmunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al., 1983;Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al.,1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.;1990 Immunoglobulin Light Chain Queen et al., 1983; Picard et al., 1984T-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo et al.;1990 HLA DQ a and/or DQ β Sullivan et al., 1987 β-Interferon Goodbournet al., 1986; Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin etal., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-DRa Shermanet al., 1989 β-Actin Kawamoto et al., 1988; Ng et al.; 1989 MuscleCreatine Kinase (MCK) Jaynes et al., 1988; Horlick et al., 1989; Johnsonet al., 1989 Prealbumin (Transthyretin) Costa et al., 1988 Elastase IOmitz et al., 1987 Metallothionein (MTII) Karin et al., 1987; Culotta etal., 1989 Collagenase Pinkert et al., 1987; Angel et al., 1987 AlbuminPinkert et al., 1987; Tronche et al., 1989, 1990 α-Fetoprotein Godboutet al., 1988; Campere et al., 1989 t-Globin Bodine et al., 1987;Perez-Stable et al., 1990 β-Globin Trudel et al., 1987 c-fos Cohen etal., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 Insulin Edlundet al., 1985 Neural Cell Adhesion Molecule Hirsh et al., 1990 (NCAM)α₁-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwang et al.,1990 Mouse and/or Type I Collagen Ripe et al., 1989 Glucose-RegulatedProteins Chang et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsenet al., 1986 Human Serum Amyloid A (SAA) Edbrooke et al., 1989 TroponinI (TN I) Yutzey et al., 1989 Platelet-Derived Growth Factor Pech et al.,1989 (PDGF) Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerjiet al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al.,1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wanget al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al.,1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinkaet al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; deVilliers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbelland/or Villarreal, 1988 Retroviruses Kriegler et al., 1982, 1983;Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze etal., 1986; Miksicek et al., 1986; Celander et al., 1987; Thiesen et al.,1988; Celander et al., 1988; Chol et al., 1988; Reisman et al., 1989Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos and/orWilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al.,1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987;Glue et al., 1988 Hepatitis B Virus Bulla et al., 1986; Jameel et al.,1986; Shaul et al., 1987; Spandau et al., 1988; Vannice et al., 1988Human Immunodeficiency Virus Muesing et al., 1987; Hauber et al., 1988;Jakobovits et al., 1988; Feng et al., 1988; Takebe et al., 1988; Rosenet al., 1988; Berkhout et al., 1989; Laspia et al., 1989; Sharp et al.,1989; Braddock et al., 1989 Cytomegalovirus (CMV) Weber et al., 1984;Boshart et al., 1985; Foecking et al., 1986 Gibbon Ape Leukemia VirusHolbrook et al., 1987; Quinn et al., 1989

TABLE 4 Inducible Elements Element Inducer References MT II PhorbolEster (TFA) Palmiter et al., 1982; Haslinger et Heavy metals al., 1985;Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin etal., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouse mammaryGlucocorticoids Huang et al., 1981; Lee et al., tumor virus) 1981;Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984; Ponta etal., 1985; Sakai et al., 1988 β-Interferon poly(rI)x Tavernier et al.,1983 poly(rc) Adenovirus 5 E2 E1A Imperiale et al., 1984 CollagenasePhorbol Ester (TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA)Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al., 1987b MurineMX Gene Interferon, Newcastle Hug et al., 1988 Disease Virus GRP78 GeneA23187 Resendez et al., 1988 α-2-Macroglobulin IL-6 Kunz et al., 1989Vimentin Serum Rittling et al., 1989 MHC Class I Gene H-2κb InterferonBlanar et al., 1989 HSP70 E1A, SV40 Large T Taylor et al., 1989, 1990a,1990b Antigen Proliferin Phorbol Ester-TPA Mordacq et al., 1989 TumorNecrosis Factor PMA Hensel et al., 1989 Thyroid Stimulating ThyroidHormone Chatterjee et al., 1989 Hormone α Gene

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Nonlimiting examples of such regions include the human LIMK2 gene(Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et al.,1998), murine epididymal retinoic acid-binding gene (Lareyre et al.,1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen(Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee, et al., 1997),insulin-like growth factor II (Wu et al., 1997), and human plateletendothelial cell adhesion molecule-1 (Almendro et al., 1996).

2. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, each herein incorporated by reference).

3. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector (see, for example, Carbonelli et al., 1999, Levensonet al., 1998, and Cocea, 1997, incorporated herein by reference.)“Restriction enzyme digestion” refers to catalytic cleavage of a nucleicacid molecule with an enzyme that functions only at specific locationsin a nucleic acid molecule. Many of these restriction enzymes arecommercially available. Use of such enzymes is widely understood bythose of skill in the art. Frequently, a vector is linearized orfragmented using a restriction enzyme that cuts within the MCS to enableexogenous sequences to be ligated to the vector. “Ligation” refers tothe process of forming phosphodiester bonds between two nucleic acidfragments, which may or may not be contiguous with each other.Techniques involving restriction enzymes and ligation reactions are wellknown to those of skill in the art of recombinant technology.

4. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (see,for example, Chandler et al., 1997, herein incorporated by reference.)

5. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

6. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal or the bovine growth hormone polyadenylationsignal, convenient and known to function well in various target cells.Polyadenylation may increase the stability of the transcript or mayfacilitate cytoplasmic transport.

7. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

8. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

9. Plasmid Vectors

In certain embodiments, a plasmid vector is contemplated for use totransform a host cell. In general, plasmid vectors containing repliconand control sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries a replication site, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. In anon-limiting example, E. coli is often transformed using derivatives ofpBR322, a plasmid derived from an E. coli species. pBR322 contains genesfor ampicillin and tetracycline resistance and thus provides easy meansfor identifying transformed cells. The pBR plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, forexample, promoters which can be used by the microbial organism forexpression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example, thephage lambda GEMTMλ11 may be utilized in making a recombinant phagevector which can be used to transform host cells, such as, for example,E. coli LE392.

Further useful plasmid vectors include pIN vectors (Inouye et al.,1985); and pGEX vectors, for use in generating glutathione S-transferase(GST) soluble fusion proteins for later purification and separation orcleavage. Other suitable fusion proteins are those with β-galactosidase,ubiquitin, and the like.

Bacterial host cells, for example, E. coli, comprising the expressionvector, are grown in any of a number of suitable media, for example, LB.The expression of the recombinant protein in certain vectors may beinduced, as would be understood by those of skill in the art, bycontacting a host cell with an agent specific for certain promoters,e.g, by adding IPTG to the media or by switching incubation to a highertemperature. After culturing the bacteria for a further period,generally of between 2 and 24 h, the cells are collected bycentrifugation and washed to remove residual media.

10. Viral Vectors

The ability of certain viruses to infect cells or enter cells viareceptor-mediated endocytosis, and to integrate into host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g,mammalian cells). Vaccine components of the present invention may be aviral vector that encode one or more folate binding protein variantantigenic compositions or other components such as, for example, afolate binding protein variant immunomodulator or adjuvant. Non-limitingexamples of virus vectors that may be used to deliver a nucleic acid ofthe present invention are described below.

a. Adenoviral Vectors

A particular method for delivery of the nucleic acid involves the use ofan adenovirus expression vector. Although adenovirus vectors are knownto have a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to ultimately express a tissue orcell-specific construct that has been cloned therein. Knowledge of thegenetic organization or adenovirus, a 36 kb, linear, double-stranded DNAvirus, allows substitution of large pieces of adenoviral DNA withforeign sequences up to 7 kb (Grunhaus and Horwitz, 1992).

b. AAV Vectors

The nucleic acid may be introduced into the cell using adenovirusassisted transfection. Increased transfection efficiencies have beenreported in cell systems using adenovirus coupled systems (Kelleher andVos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno-associated virus(AAV) is an attractive vector system for use in the folate bindingprotein variant vaccines of the present invention as it has a highfrequency of integration and it can infect nondividing cells, thusmaking it useful for delivery of genes into mammalian cells, forexample, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a broadhost range for infectivity (Tratschin et al., 1984; Laughlin et al.,1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Detailsconcerning the generation and use of rAAV vectors are described in U.S.Pat. Nos. 5,139,941 and 4,797,368, each incorporated herein byreference.

c. Retroviral Vectors

Retroviruses have promise as folate binding protein variant antigendelivery vectors in vaccines due to their ability to integrate theirgenes into the host genome, transferring a large amount of foreigngenetic material, infecting a broad spectrum of species and cell typesand of being packaged in special cell lines (Miller, 1992).

In order to construct a folate binding protein variant vaccineretroviral vector, a nucleic acid (e.g, one encoding an folate bindingprotein variant antigen of interest) is inserted into the viral genomein the place of certain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into a special cell line (e.g, bycalcium phosphate precipitation for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media (Nicolasand Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are able toinfect a broad variety of cell types. However, integration and stableexpression require the division of host cells (Paskind et al., 1975).

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Lentiviral vectors are well known in the art(see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomeret al., 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples oflentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 andthe Simian Immunodeficiency Virus: SIV. Lentiviral vectors have beengenerated by multiply attenuating the HIV virulence genes, for example,the genes env, vif, vpr, vpu and nef are deleted making the vectorbiologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. For example, recombinantlentivirus capable of infecting a non-dividing cell wherein a suitablehost cell is transfected with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat at isdescribed in U.S. Pat. No. 5,994,136, incorporated herein by reference.One may target the recombinant virus by linkage of the envelope proteinwith an antibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including a regulatoryregion) of interest into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target-specific.

d. Other Viral Vectors

Other viral vectors may be employed as vaccine constructs in the presentinvention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988),sindbis virus, cytomegalovirus and herpes simplex virus may be employed.They offer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

e. Vaccine Delivery Using Modified Viruses

A nucleic acid to be delivered may be housed within an infective virusthat has been engineered to express a specific binding ligand. The virusparticle will thus bind specifically to the cognate receptors of thetarget cell and deliver the contents to the cell. A novel approachdesigned to allow specific targeting of retrovirus vectors was recentlydeveloped based on the chemical modification of a retrovirus by thechemical addition of lactose residues to the viral envelope. Thismodification can permit the specific infection of hepatocytes viasialoglycoprotein receptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et al.,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux et al., 1989). Thus, it is contemplated that antibodies,specific binding ligands and/or other targeting moieties may be used tospecifically transfect APC types.

11. Vector Delivery and Cell Transformation

Suitable methods for nucleic acid delivery for transformation of anorganelle, a cell, a tissue or an organism for use with the currentinvention are believed to include virtually any method by which anucleic acid (e.g, DNA) can be introduced into an organelle, a cell, atissue or an organism, as described herein or as would be known to oneof ordinary skill in the art. Such methods include, but are not limitedto, direct delivery of DNA such as by injection (U.S. Pat. Nos.5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932,5,656,610, 5,589,466 and 5,580,859, each incorporated herein byreference), including microinjection (Harlan and Weintraub, 1985; U.S.Pat. No. 5,789,215, incorporated herein by reference); byelectroporation (U.S. Pat. No. 5,384,253, incorporated herein byreference; Tur-Kaspa et al., 1986; Potter et al., 1984); by calciumphosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama,1987; Rippe et al., 1990); by using DEAE-dextran followed bypolyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimeret al., 1987); by liposome mediated transfection (Nicolau and Sene,1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980;Kaneda et al., 1989; Kato et al., 1991) and receptor-mediatedtransfection (Wu and Wu, 1987; Wu and Wu, 1988); by microprojectilebombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat.Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880,and each incorporated herein by reference); by agitation with siliconcarbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and5,464,765, each incorporated herein by reference); byAgrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); or by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), andany combination of such methods. Through the application of techniquessuch as these, organelle(s), cell(s), tissue(s) or organism(s) may bestably or transiently transformed.

a. Injection

In certain embodiments, a nucleic acid may be delivered to an organelle,a cell, a tissue or an organism via one or more injections (i.e., aneedle injection). Methods of injection of nucleic acids are describedherein, and are well known to those of ordinary skill in the art.Further embodiments of the present invention include the introduction ofa nucleic acid by direct microinjection to a cell. Direct microinjectionhas been used to introduce nucleic acid constructs into Xenopus oocytes(Harland and Weintraub, 1985). The amount of folate binding proteinvariant used may vary upon the nature of the antigen as well as theorganelle, cell, tissue or organism used

b. Electroporation

In certain embodiments of the present invention, a nucleic acid isintroduced into an organelle, a cell, a tissue or an organism viaelectroporation. Electroporation involves the exposure of a suspensionof cells and DNA to a high-voltage electric discharge. In some variantsof this method, certain cell wall-degrading enzymes, such aspectin-degrading enzymes, are employed to render the target recipientcells more susceptible to transformation by electroporation thanuntreated cells (U.S. Pat. No. 5,384,253, incorporated herein byreference). Alternatively, recipient cells can be made more susceptibleto transformation by mechanical wounding.

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre-B lymphocytes have been transfected with humankappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocyteshave been transfected with the chloramphenicol acetyltransferase gene(Tur-Kaspa et al., 1986) in this manner.

To effect transformation by electroporation in cells such as, forexample, plant cells, one may employ either friable tissues, such as asuspension culture of cells or embryogenic callus or alternatively onemay transform immature embryos or other organized tissue directly. Inthis technique, one would partially degrade the cell walls of the chosencells by exposing them to pectin-degrading enzymes (pectolyases) ormechanically wounding in a controlled manner. Examples of some specieswhich have been transformed by electroporation of intact cells includemaize (U.S. Pat. No. 5,384,253; Rhodes et al., 1995; D'Halluin et al.,1992), wheat (Zhou et al., 1993), tomato (Hou and Lin, 1996), soybean(Christou et al., 1987) and tobacco (Lee et al., 1989).

One also may employ protoplasts for electroporation transformation ofplant cells (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon-derivedprotoplasts is described by Dhir and Widholm in International PatentApplication No. WO 9217598, incorporated herein by reference. Otherexamples of species for which protoplast transformation has beendescribed include barley (Lazerri, 1995), sorghum (Battraw et al.,1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) andtomato (Tsukada, 1989).

c. Calcium Phosphate

In other embodiments of the present invention, a nucleic acid isintroduced to the cells using calcium phosphate precipitation. Human KBcells have been transfected with adenovirus 5 DNA (Graham and Van DerEb, 1973) using this technique. Also in this manner, mouse L(A9), mouseC127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with aneomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes weretransfected with a variety of marker genes (Rippe et al., 1990).

d. DEAE-Dextran

In another embodiment, a nucleic acid is delivered into a cell usingDEAE-dextran followed by polyethylene glycol. In this manner, reporterplasmids were introduced into mouse myeloma and erythroleukemia cells(Gopal, 1985).

e. Liposome-Mediated Transfection

In a further embodiment of the invention, one or more vaccine componentsor nucleic acids may be entrapped in a lipid complex such as, forexample, a liposome. Liposomes are vesicular structures characterized bya phospholipid bilayer membrane and an inner aqueous medium.Multilamellar liposomes have multiple lipid layers separated by aqueousmedium. They form spontaneously when phospholipids are suspended in anexcess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated is an nucleic acid complexed withLipofectamine (Gibco BRL) or Superfect (Qiagen).

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful (Nicolau and Sene, 1982; Fraley et al.,1979; Nicolau et al., 1987). The feasibility of liposome-mediateddelivery and expression of foreign DNA in cultured chick embryo, HeLaand hepatoma cells has also been demonstrated (Wong et al., 1980).

In certain embodiments of the invention, a liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, aliposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, a liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In other embodiments, a deliveryvehicle may comprise a ligand and a liposome.

f. Receptor Mediated Transfection

One or more vaccine components or nucleic acids, may be employed todelivered using a receptor-mediated delivery vehicle. These takeadvantage of the selective uptake of macromolecules by receptor-mediatedendocytosis that will be occurring in the target cells. In view of thecell type-specific distribution of various receptors, this deliverymethod adds another degree of specificity to the present invention.Specific delivery in the context of another mammalian cell type has beendescribed (Wu and Wu, 1993, incorporated herein by reference).

Certain receptor-mediated gene targeting vehicles comprise a cellreceptor-specific ligand and a nucleic acid-binding agent. Otherscomprise a cell receptor-specific ligand to which the nucleic acid to bedelivered has been operatively attached. Several ligands have been usedfor receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al.,1990; Perales et al., 1994; Myers, EPO 0273085), which establishes theoperability of the technique. Specific delivery in the context ofanother mammalian cell type has been described (Wu and Wu, 1993;incorporated herein by reference). In certain aspects of the presentinvention, a ligand will be chosen to correspond to a receptorspecifically expressed on the target cell population.

In other embodiments, a nucleic acid delivery vehicle component of acell-specific nucleic acid targeting vehicle may comprise a specificbinding ligand in combination with a liposome. The nucleic acid(s) to bedelivered are housed within the liposome and the specific binding ligandis functionally incorporated into the liposome membrane. The liposomewill thus specifically bind to the receptor(s) of a target cell anddeliver the contents to a cell. Such systems have been shown to befunctional using systems in which, for example, epidermal growth factor(EGF) is used in the receptor-mediated delivery of a nucleic acid tocells that exhibit upregulation of the EGF receptor.

In still further embodiments, the nucleic acid delivery vehiclecomponent of a targeted delivery vehicle may be a liposome itself, whichwill preferably comprise one or more lipids or glycoproteins that directcell-specific binding. For example, lactosyl-ceramide, agalactose-terminal asialganglioside, have been incorporated intoliposomes and observed an increase in the uptake of the insulin gene byhepatocytes (Nicolau et al., 1987). It is contemplated that thetissue-specific transforming constructs of the present invention can bespecifically delivered into a target cell in a similar manner.

g. Microprojectile Bombardment

Microprojectile bombardment techniques can be used to introduce anucleic acid into at least one, organelle, cell, tissue or organism(U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No.5,610,042; and PCT Application WO 94/09699; each of which isincorporated herein by reference). This method depends on the ability toaccelerate DNA-coated microprojectiles to a high velocity allowing themto pierce cell membranes and enter cells without killing them (Klein etal., 1987). There are a wide variety of microprojectile bombardmenttechniques known in the art, many of which are applicable to theinvention.

Microprojectile bombardment may be used to transform various cell(s),tissue(s) or organism(s), such as for example any plant species.Examples of species which have been transformed by microprojectilebombardment include monocot species such as maize (PCT Application WO95/06128), barley (Ritala et al., 1994; Hensgens et al., 1993), wheat(U.S. Pat. No. 5,563,055, incorporated herein by reference), rice(Hensgens et al., 1993), oat (Torbet et al., 1995; Torbet et al., 1998),rye (Hensgens et al., 1993), sugarcane (Bower et al., 1992), and sorghum(Casas et al., 1993; Hagio et al., 1991); as well as a number of dicotsincluding tobacco (Tomes et al., 1990; Buising and Benbow, 1994),soybean (U.S. Pat. No. 5,322,783, incorporated herein by reference),sunflower (Knittel et al. 1994), peanut (Singsit et al., 1997), cotton(McCabe and Martinell, 1993), tomato (VanEck et al. 1995), and legumesin general (U.S. Pat. No. 5,563,055, incorporated herein by reference).

In this microprojectile bombardment, one or more particles may be coatedwith at least one nucleic acid and delivered into cells by a propellingforce. Several devices for accelerating small particles have beendeveloped. One such device relies on a high voltage discharge togenerate an electrical current, which in turn provides the motive force(Yang et al., 1990). The microprojectiles used have consisted ofbiologically inert substances such as tungsten or gold particles orbeads. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. It is contemplated that in someinstances DNA precipitation onto metal particles would not be necessaryfor DNA delivery to a recipient cell using microprojectile bombardment.However, it is contemplated that particles may contain DNA rather thanbe coated with DNA. DNA-coated particles may increase the level of DNAdelivery via particle bombardment but are not, in and of themselves,necessary.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells may be arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate.

12. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organisms that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny. Asused herein, the terms “engineered” and “recombinant” cells or hostcells are intended to refer to a cell into which an exogenous nucleicacid sequence, such as, for example, a vector, has been introduced.Therefore, recombinant cells are distinguishable from naturallyoccurring cells which do not contain a recombinantly introduced nucleicacid.

In certain embodiments, it is contemplated that RNAs or proteinaceoussequences may be co-expressed with other selected RNAs or proteinaceoussequences in the same host cell. Co-expression may be achieved byco-transfecting the host cell with two or more distinct recombinantvectors. Alternatively, a single recombinant vector may be constructedto include multiple distinct coding regions for RNAs, which could thenbe expressed in host cells transfected with the single vector.

A tissue may comprise a host cell or cells to be transformed with afolate binding protein variant. The tissue may be part or separated froman organism. In certain embodiments, a tissue may comprise, but is notlimited to, adipocytes, alveolar, ameloblasts, axon, basal cells, blood(e.g, lymphocytes), blood vessel, bone, bone marrow, brain, breast,cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial,epithelial, esophagus, facia, fibroblast, follicular, ganglion cells,glial cells, goblet cells, kidney, liver, lung, lymph node, muscle,neuron, ovaries, pancreas, peripheral blood, prostate, skin, skin, smallintestine, spleen, stem cells, stomach, testes, anthers, ascite tissue,cobs, ears, flowers, husks, kernels, leaves, meristematic cells, pollen,root tips, roots, silk, stalks, and all cancers thereof.

In certain embodiments, the host cell or tissue may be comprised in atleast one organism. In certain embodiments, the organism may be, but isnot limited to, a prokayote (e.g, a eubacteria, an archaea) or aneukaryote, as would be understood by one of ordinary skill in the art.

Numerous cell lines and cultures are available for use as a host cell,and they can be obtained through the American Type Culture Collection(ATCC), which is an organization that serves as an archive for livingcultures and genetic materials. An appropriate host can be determined byone of skill in the art based on the vector backbone and the desiredresult. A plasmid or cosmid, for example, can be introduced into aprokaryote host cell for replication of many vectors. Cell typesavailable for vector replication and/or expressioninclude, but are notlimited to, bacteria, such as E. coli (e.g, E. coli strain RR1, E. coliLE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as well as E. coliW3110 (F′, lambda, prototrophic, ATCC No. 273325), bacilli such asBacillus subtilis; and other enterobacteriaceae such as Salmonellatyphimurium, Serratia marcescens, various Pseudomonas specie, DHSa,JM109, and KCB, as well as a number of commercially available bacterialhosts such as SURE.®. Competent Cells and SOLOPACKa Gold Cells(STRATAGENE®, La Jolla). In certain embodiments, bacterial cells such asE. coli LE392 are particularly contemplated as host cells for phageviruses.

Examples of eukaryotic host cells for replication and/or expression of avector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, Cos,CHO, Saos, and PC12. Many host cells from various cell types andorganisms are available and would be known to one of skill in the art.Similarly, a viral vector may be used in conjunction with either aeukaryotic or prokaryotic host cell, particularly one that is permissivefor replication or expression of the vector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

13. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

Other examples of expression systems include STRATAGENE®'s COMPLETECONTROLä. Inducible Mammalian Expression System, which involves asynthetic ecdysone-inducible receptor, or its pET Expression System, anE. coli expression system. Another example of an inducible expressionsystem is available from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

It is contemplated that the proteins, polypeptides or peptides producedby the methods of the invention may be “overexpressed”, i.e., expressedin increased levels relative to its natural expression in cells. Suchoverexpression may be assessed by a variety of methods, includingradiolabeling and/or protein purification. However, simple and directmethods are preferred, for example, those involving SDS/PAGE and proteinstaining or western blotting, followed by quantitative analyses, such asdensitometric scanning of the resultant gel or blot. A specific increasein the level of the recombinant protein, polypeptide or peptide incomparison to the level in natural cells is indicative ofoverexpression, as is a relative abundance of the specific protein,polypeptides or peptides in relation to the other proteins produced bythe host cell and, e.g, visible on a gel.

In some embodiments, the expressed proteinaceous sequence forms aninclusion body in the host cell, the host cells are lysed, for example,by disruption in a cell homogenizer, washed and/or centrifuged toseparate the dense inclusion bodies and cell membranes from the solublecell components. This centrifugation can be performed under conditionswhereby the dense inclusion bodies are selectively enriched byincorporation of sugars, such as sucrose, into the buffer andcentrifugation at a selective speed. Inclusion bodies may be solubilizedin solutions containing high concentrations of urea (e.g 8M) orchaotropic agents such as guanidine hydrochloride in the presence ofreducing agents, such as β-mercaptoethanol or DTT (dithiothreitol), andrefolded into a more desirable conformation, as would be known to one ofordinary skill in the art.

G. Vaccine Component Purification

In any case, a vaccine component (e.g, an antigenic peptide orpolypeptide or nucleic acid encoding a proteinaceous composition) may beisolated and/or purified from the chemical synthesis reagents, cell orcellular components. In a method of producing the vaccine component,purification is accomplished by any appropriate technique that isdescribed herein or well known to those of skill in the art (e.g,Sambrook et al., 1987). Although preferred for use in certainembodiments, there is no general requirement that an antigeniccomposition of the present invention or other vaccine component alwaysbe provided in their most purified state. Indeed, it is contemplatedthat a less substantially purified vaccine component, which isnonetheless enriched in the desired compound, relative to the naturalstate, will have utility in certain embodiments, such as, for example,total recovery of protein product, or in maintaining the activity of anexpressed protein. However, it is contemplate that inactive productsalso have utility in certain embodiments, such as, e.g, in determiningantigenicity via antibody generation.

The present invention also provides purified, and in preferredembodiments, substantially purified vaccines or vaccine components. Theterm “purified vaccine component” as used herein, is intended to referto at least one vaccine component (e.g, a proteinaceous composition,isolatable from cells), wherein the component is purified to any degreerelative to its naturally-obtainable state, e.g, relative to its puritywithin a cellular extract or reagents of chemical synthesis. In certainaspects wherein the vaccine component is a proteinaceous composition, apurified vaccine component also refers to a wild-type or mutant protein,polypeptide, or peptide free from the environment in which it naturallyoccurs.

Where the term “substantially purified” is used, this will refer to acomposition in which the specific compound (e.g, a protein, polypeptide,or peptide) forms the major component of the composition, such asconstituting about 50% of the compounds in the composition or more. Inpreferred embodiments, a substantially purified vaccine component willconstitute more than about 60%, about 70%, about 80%, about 90%, about95%, about 99% or even more of the compounds in the composition.

In certain embodiments, a vaccine component may be purified tohomogeneity. As applied to the present invention, “purified tohomogeneity,” means that the vaccine component has a level of puritywhere the compound is substantially free from other chemicals,biomolecules or cells. For example, a purified peptide, polypeptide orprotein will often be sufficiently free of other protein components sothat degradative sequencing may be performed successfully. Variousmethods for quantifying the degree of purification of a vaccinecomponent will be known to those of skill in the art in light of thepresent disclosure. These include, for example, determining the specificprotein activity of a fraction (e.g, antigenicity), or assessing thenumber of polypeptides within a fraction by gel electrophoresis.

Various techniques suitable for use in chemical, biomolecule orbiological purification, well known to those of skill in the art, may beapplicable to preparation of a vaccine component of the presentinvention. These include, for example, precipitation with ammoniumsulfate, PEG, antibodies and the like or by heat denaturation, followedby centrifugation; fractionation, chromatographic procedures, includingbut not limited to, partition chromatograph (e.g, paper chromatograph,thin-layer chromatograph (TLC), gas-liquid chromatography and gelchromatography) gas chromatography, high performance liquidchromatography, affinity chromatography, supercritical flowchromatography ion exchange, gel filtration, reverse phase,hydroxylapatite, lectin affinity; isoelectric focusing and gelelectrophoresis (see for example, Sambrook et al. 1989; and Freifelder,Physical Biochemistry, Second Edition, pages 238-246, incorporatedherein by reference).

Given many DNA and proteins are known (see for example, the NationalCenter for Biotechnology Information's Genbank® and GenPept databases),or may be identified and amplified using the methods described herein,any purification method for recombinately expressed nucleic acid orproteinaceous sequences known to those of skill in the art can now beemployed. In certain aspects, a nucleic acid may be purified onpolyacrylamide gels, and/or cesium chloride centrifugation gradients, orby any other means known to one of ordinary skill in the art (see forexample, Sambrook et al. 1989, incorporated herein by reference). Infurther aspects, a purification of a proteinaceous sequence may beconducted by recombinately expressing the sequence as a fusion protein.Such purification methods are routine in the art. This is exemplified bythe generation of an specific protein-glutathione S-transferase fusionprotein, expression in E. coli, and isolation to homogeneity usingaffinity chromatography on glutathione-agarose or the generation of apolyhistidine tag on the N- or C-terminus of the protein, and subsequentpurification using Ni-affinity chromatography. In particular aspects,cells or other components of the vaccine may be purified by flowcytometry. Flow cytometry involves the separation of cells or otherparticles in a liquid sample, and is well known in the art (see, forexample, U.S. Pat. Nos. 3,826,364, 4,284,412, 4,989,977, 4,498,766,5,478,722, 4,857,451, 4,774,189, 4,767,206, 4,714,682, 5,160,974 and4,661,913). Any of these techniques described herein, and combinationsof these and any other techniques known to skilled artisans, may be usedto purify and/or assay the purity of the various chemicals,proteinaceous compounds, nucleic acids, cellular materials and/or cellsthat may comprise a vaccine of the present invention. As is generallyknown in the art, it is believed that the order of conducting thevarious purification steps may be changed, or that certain steps may beomitted, and still result in a suitable method for the preparation of asubstantially purified antigen or other vaccine component.

H. Additional Vaccine Components

It is contemplated that an antigenic composition of the invention may becombined with one or more additional components to form a more effectivevaccine. Non-limiting examples of additional components include, forexample, one or more additional antigens, immunomodulators or adjuvantsto stimulate an immune response to an antigenic composition of thepresent invention and/or the additional component(s).

1. Immunomodulators

For example, it is contemplated that immunomodulators can be included inthe vaccine to augment a cell's or a patient's (e.g, an animal's)response. Immunomodulators can be included as purified proteins, nucleicacids encoding immunomodulators, and/or cells that expressimmunomodulators in the vaccine composition. The following sections listnon-limiting examples of immunomodulators that are of interest, and itis contemplated that various combinations of immunomodulators may beused in certain embodiments (e.g, a cytokine and a chemokine).

In another aspects of the invention, it is contemplated that the folatebinding protein variant composition may further comprise atherapeutically effective composition of an immunomodulator. It isenvisioned that an immunomodulator would constitute a cytokine,hematapoietin, colony stimulating factor, interleukin, interferon,growth factor or combination thereof. As used herein certainembodiments, the terms “cytokine” are the same as described in U.S. Pat.No. 5,851,984, incorporated herein by reference in its entirety, whichreads in relevant part:

“The term ‘cytokine’ is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, growth factorsand traditional polypeptide hormones. Included among the cytokines aregrowth hormones such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-.alpha. and -.beta.; mullerian-inhibitingsubstance; mouse gonadotropin-associated peptide; inhibin; activin;vascular endothelial growth factor; integrin; thrombopoietin (TPO);nerve growth factors such as NGF-.beta.; platelet-growth factor;transforming growth factors (TGFs) such as TGF-.alpha. and TGF-.beta.;insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-a, -.b, and -g;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, LIF, G-CSF, GM-CSF, M-CSF, EPO, kit-ligand or FLT-3. As usedherein, the term cytokine includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokines.

a. β-interferon

β-interferon (IFN-b) is low molecular weight protein that is produced bymany cell types, including epithelial cells, fibroblasts andmacrophages. Cells that express endogenous IFN-b are resistant to viralinfection and replication. The b-interferon genes from mouse (GenBank®accession numbers X14455, X14029) and human (GenBank® accession numbersJ00218, K00616 and M11029) have been isolated and sequenced. IFN-b is amultifunctional glycoprotein that can inhibit tumor growth bothdirectly, by suppressing cell replication and inducing differentiationor apoptosis and indirectly by activating tumoricidal properties ofmacrophages and NK cells, by suppressing tumor angiogenesis and bystimulating specific immune response.

b. Interleukin-2

Interleukin-2 (IL-2), originally designated T-cell growth factor I, is ahighly proficient inducer of T-cell proliferation and is a growth factorfor all subpopulations of T-lymphocytes. IL-2 is an antigen independentproliferation factor that induces cell cycle progression in restingcells and thus allows clonal expansion of activated T-lymphocytes. Sincefreshly isolated leukemic cells also secrete IL2 and respond to it IL2may function as an autocrine growth modulator for these cells capable ofworsening ATL. IL2 also promotes the proliferation of activated B-cellsalthough this requires the presence of additional factors, for example,IL4. In vitro IL2 also stimulates the growth of oligodendroglial cells.Due to its effects on T-cells and B-cells IL2 is a central regulator ofimmune responses. It also plays a role in anti-inflammatory reactions,in hematopoiesis and in tumor surveillance. IL-2 stimulates thesynthesis of IFN-g in peripheral leukocytes and also induces thesecretion of IL-1, TNF-a and TNF-b. The induction of the secretion oftumoricidal cytokines, apart from the activity in the expansion of LAKcells, (lymphokine-activated killer cells) are probably the main factorsresponsible for the antitumor activity of IL2.

c. GM-CSF

GM-CSF stimulates the proliferation and differentiation of neutrophilic,eosinophilic, and monocytic lineages. It also functionally activates thecorresponding mature forms, enhancing, for example, to the expression ofcertain cell surface adhesion proteins (CD-11A, CD-11C). Theoverexpression of these proteins could be one explanation for theobserved local accumulation of granulocytes at sites of inflammation. Inaddition, GM-CSF also enhances expression of receptors for fMLP(Formyl-Met-Leu-Phe) which is a stimulator of neutrophil activity.

d. Cytokines

Interleukins, cytokines, nucleic acids encoding interleukins orcytokines, and/or cells expressing such compounds are contemplated aspossible vaccine components. Interleukins and cytokines, include but arenot limited to interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-18,β-interferon, α-interferon, γ-interferon, angiostatin, thrombospondin,endostatin, GM-CSF, G-CSF, M-CSF, METH-1, METH-2, tumor necrosis factor,TGFb, LT and combinations thereof.

e. Chemokines

Chemokines, nucleic acids that encode for chemokines, and/or cells thatexpress such also may be used as vaccine components. Chemokinesgenerally act as chemoattractants to recruit immune effector cells tothe site of chemokine expression. It may be advantageous to express aparticular chemokine coding sequence in combination with, for example, acytokine coding sequence, to enhance the recruitment of other immunesystem components to the site of treatment. Such chemokines include, forexample, RANTES, MCAF, MIP1-alpha, MIP1-Beta, IP-10 and combinationsthereof. The skilled artisan will recognize that certain cytokines arealso known to have chemoattractant effects and could also be classifiedunder the term chemokines.

f. Immunogenic Carrier Proteins

In certain embodiments, an antigenic composition's may be chemicallycoupled to a carrier or recombinantly expressed with a immunogeniccarrier peptide or polypetide (e.g, a antigen-carrier fusion peptide orpolypeptide) to enhance an immune reaction. Exemplary and preferredimmunogenic carrier amino acid sequences include hepatitis B surfaceantigen, keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).Other albumins such as ovalbumin, mouse serum albumin or rabbit serumalbumin also can be used as immunogenic carrier proteins. Means forconjugating a polypeptide or peptide to a immunogenic carrier proteinare well known in the art and include, for example, glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

g. Biological Response Modifiers

It may be desirable to coadminister biologic response modifiers (BRM),which have been shown to upregulate T cell immunity or downregulatesuppressor cell activity. Such BRMs include, but are not limited to,cimetidine (CIM; 1200 mg/d) (Smith/Kline, Pa.); low-dosecyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, N.J.), or a geneencoding a protein involved in one or more immune helper functions, suchas B-7.

2. Adjuvants

Immunization protocols have used adjuvants to stimulate responses formany years, and as such adjuvants are well known to one of ordinaryskill in the art. Some adjuvants affect the way in which antigens arepresented. For example, the immune response is increased when proteinantigens are precipitated by alum. Emulsification of antigens alsoprolongs the duration of antigen presentation.

In one aspect, an adjuvant effect is achieved by use of an agent such asalum used in about 0.05 to about 0.1% solution in phosphate bufferedsaline. Alternatively, the antigen is made as an admixture withsynthetic polymers of sugars (Carbopol®) used as an about 0.25%solution. Adjuvant effect may also be made my aggregation of the antigenin the vaccine by heat treatment with temperatures ranging between about70° to about 101° C. for a 30-second to 2-minute period, respectively.Aggregation by reactivating with pepsin treated (Fab) antibodies toalbumin, mixture with bacterial cell(s) such as C. parvum or anendotoxin or a lipopolysaccharide components of Gram-negative bacteria,emulsion in physiologically acceptable oil vehicles such as mannidemono-oleate (Aracel A) or emulsion with a 20% solution of aperfluorocarbon (Fluosol-DA®) used as a block substitute also may beemployed.

Some adjuvants, for example, are certain organic molecules obtained frombacteria, act on the host rather than on the antigen. An example ismuramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine [MDP]), abacterial peptidoglycan. The effects of MDP, as with most adjuvants, arenot fully understood. MDP stimulates macrophages but also appears tostimulate B cells directly. The effects of adjuvants, therefore, are notantigen-specific. If they are administered together with a purifiedantigen, however, they can be used to selectively promote the responseto the antigen.

Adjuvants have been used experimentally to promote a generalizedincrease in immunity against unknown antigens (e.g, U.S. Pat. No.4,877,611). This has been attempted particularly in the treatment ofcancer. For many cancers, there is compelling evidence that the immunesystem participates in host defense against the tumor cells, but only afraction of the likely total number of tumor-specific antigens arebelieved to have been identified to date. However, using the presentinvention, the inclusion of a suitable adjuvant into the membrane of anirradiated tumor cell will likely increase the anti-tumor responseirrespective of the molecular identification of the prominent antigens.This is a particularly important and time-saving feature of theinvention.

In certain embodiments, hemocyanins and hemoerythrins may also be usedin the invention. The use of hemocyanin from keyhole limpet (KLH) ispreferred in certain embodiments, although other molluscan and arthropodhemocyanins and hemoerythrins may be employed.

Various polysaccharide adjuvants may also be used. For example, the useof various pneumococcal polysaccharide adjuvants on the antibodyresponses of mice has been described (Yin et al., 1989). The doses thatproduce optimal responses, or that otherwise do not produce suppression,should be employed as indicated (Yin et al., 1989). Polyamine varietiesof polysaccharides are particularly preferred, such as chitin andchitosan, including deacetylated chitin.

Another group of adjuvants are the muramyl dipeptide (MDP,N-acetylmuramyl-L-alanyl-D-isoglutamine) group of bacterialpeptidoglycans. Derivatives of muramyl dipeptide, such as the amino acidderivative threonyl-MDP, and the fatty acid derivative MTPPE, are alsocontemplated.

U.S. Pat. No. 4,950,645 describes a lipophilic disaccharide-tripeptidederivative of muramyl dipeptide which is described for use in artificialliposomes formed from phosphatidyl choline and phosphatidyl glycerol. Itis the to be effective in activating human monocytes and destroyingtumor cells, but is non-toxic in generally high doses. The compounds ofU.S. Pat. No. 4,950,645 and PCT Patent Application WO 91/16347, arecontemplated for use with cellular carriers and other embodiments of thepresent invention.

Another adjuvant contemplated for use in the present invention is BCG.BCG (bacillus Calmette-Guerin, an attenuated strain of Mycobacterium)and BCG-cell wall skeleton (CWS) may also be used as adjuvants in theinvention, with or without trehalose dimycolate. Trehalose dimycolatemay be used itself. Trehalose dimycolate administration has been shownto correlate with augmented resistance to influenza virus infection inmice (Azuma et al., 1988). Trehalose dimycolate may be prepared asdescribed in U.S. Pat. No. 4,579,945.

BCG is an important clinical tool because of its immunostimulatoryproperties. BCG acts to stimulate the reticulo-endothelial system,activates natural killer cells and increases proliferation ofhematopoietic stem cells. Cell wall extracts of BCG have proven to haveexcellent immune adjuvant activity. Molecular genetic tools and methodsfor mycobacteria have provided the means to introduce foreign genes intoBCG (Jacobs et al., 1987; Snapper et al., 1988; Hus son et al., 1990;Martin et al., 1990).

Live BCG is an effective and safe vaccine used worldwide to preventtuberculosis. BCG and other mycobacteria are highly effective adjuvants,and the immune response to mycobacteria has been studied extensively.With nearly 2 billion immunizations, BCG has a long record of safe usein man (Luelmo, 1982; Lotte et al., 1984). It is one of the few vaccinesthat can be given at birth, it engenders long-lived immune responseswith only a single dose, and there is a worldwide distribution networkwith experience in BCG vaccination. An exemplary BCG vaccine is sold asTICE™ BCG (Organon Inc., West Orange, N.J.).

In a typical practice of the present invention, cells of Mycobacteriumbovis-BCG are grown and harvested by methods known in the art. Forexample, they may be grown as a surface pellicle on a Sauton medium orin a fermentation vessel containing the dispersed culture in a Dubosmedium (Dubos et al., 1947; Rosenthal, 1937). All the cultures areharvested after 14 days incubation at about 37° C. Cells grown as apellicle are harvested by using a platinum loop whereas those from thefermenter are harvested by centrifugation or tangential-flow filtration.The harvested cells are resuspended in an aqueous sterile buffer medium.A typical suspension contains from about 2×10¹⁰ cells/ml to about 2×10¹²cells/ml. To this bacterial suspension, a sterile solution containing aselected enzyme which will degrade the BCG cell covering material isadded. The resultant suspension is agitated such as by stirring toensure maximal dispersal of the BCG organisms. Thereafter, a moreconcentrated cell suspension is prepared and the enzyme in theconcentrate removed, typically by washing with an aqueous buffer,employing known techniques such as tangential-flow filtration. Theenzyme-free cells are adjusted to an optimal immunological concentrationwith a cryoprotectant solution, after which they are filled into vials,ampoules, etc., and lyophilized, yielding BCG vaccine, which uponreconstitution with water is ready for immunization.

Amphipathic and surface active agents, e.g, saponin and derivatives suchas QS21 (Cambridge Biotech), form yet another group of adjuvants for usewith the immunogens of the present invention. Nonionic block copolymersurfactants (Rabinovich et al., 1994; Hunter et al., 1991) may also beemployed. Oligonucleotides are another useful group of adjuvants(Yamamoto et al., 1988). Quil A and lentinen are other adjuvants thatmay be used in certain embodiments of the present invention.

One group of adjuvants preferred for use in the invention are thedetoxified endotoxins, such as the refined detoxified endotoxin of U.S.Pat. No. 4,866,034. These refined detoxified endotoxins are effective inproducing adjuvant responses in mammals. Of course, the detoxifiedendotoxins may be combined with other adjuvants to preparemulti-adjuvant-incorporated cells. For example, combination ofdetoxified endotoxins with trehalose dimycolate is particularlycontemplated, as described in U.S. Pat. No. 4,435,386. Combinations ofdetoxified endotoxins with trehalose dimycolate and endotoxicglycolipids is also contemplated (U.S. Pat. No. 4,505,899), as iscombination of detoxified endotoxins with cell wall skeleton (CWS) orCWS and trehalose dimycolate, as described in U.S. Pat. Nos. 4,436,727,4,436,728 and 4,505,900. Combinations of just CWS and trehalosedimycolate, without detoxified endotoxins, is also envisioned to beuseful, as described in U.S. Pat. No. 4,520,019.

In other embodiments, the present invention contemplates that a varietyof adjuvants may be employed in the membranes of cells, resulting in animproved immunogenic composition. The only requirement is, generally,that the adjuvant be capable of incorporation into, physical associationwith, or conjugation to, the cell membrane of the cell in question.Those of skill in the art will know the different kinds of adjuvantsthat can be conjugated to cellular vaccines in accordance with thisinvention and these include alkyl lysophosphilipids (ALP); BCG; andbiotin (including biotinylated derivatives) among others. Certainadjuvants particularly contemplated for use are the teichoic acids fromGram positive cells. These include the lipoteichoic acids (LTA), ribitolteichoic acids (RTA) and glycerol teichoic acid (GTA). Active forms oftheir synthetic counterparts may also be employed in connection with theinvention (Takada et al., 1995a).

Various adjuvants, even those that are not commonly used in humans, maystill be employed in animals, where, for example, one desires to raiseantibodies or to subsequently obtain activated T cells. The toxicity orother adverse effects that may result from either the adjuvant or thecells, e.g, as may occur using non-irradiated tumor cells, is irrelevantin such circumstances.

One group of adjuvants preferred for use in some embodiments of thepresent invention are those that can be encoded by a nucleic acid (e.g,DNA or RNA). It is contemplated that such adjuvants may be encoded in anucleic acid (e.g, an expression vector) encoding the antigen, or in aseparate vector or other construct. These nucleic acids encoding theadjuvants can be delivered directly, such as for example with lipids orliposomes.

3. Excipients, Salts and Auxiliary Substances

An antigenic composition of the present invention may be mixed with oneor more additional components (e.g, excipients, salts, etc.) which arepharmaceutically acceptable and compatible with at least one activeingredient (e.g, antigen). Suitable excipients are, for example, water,saline, dextrose, glycerol, ethanol and combinations thereof.

An antigenic composition of the present invention may be formulated intothe vaccine as a neutral or salt form. A pharmaceutically-acceptablesalt, includes the acid addition salts (formed with the free aminogroups of the peptide) and those which are formed with inorganic acidssuch as, for example, hydrochloric or phosphoric acid, or such organicacids as acetic, oxalic, tartaric, mandelic, and the like. A salt formedwith a free carboxyl group also may be derived from an inorganic basesuch as, for example, sodium, potassium, ammonium, calcium, or ferrichydroxide, and such organic bases as isopropylamine, trimethylamine,2-ethylamino ethanol, histidine, procaine, and combinations thereof.

In addition, if desired, an antigentic composition may comprise minoramounts of one or more auxiliary substances such as for example wettingor emulsifying agents, pH buffering agents, etc. which enhance theeffectiveness of the antigenic composition or vaccine.

I. Vaccine Preparations

Once produced, synthesized and/or purified, an antigen or other vaccinecomponent may be prepared as a vaccine for administration to a patient.The preparation of a vaccine is generally well understood in the art, asexemplified by U.S. Pat. Nos. 4,608,251, 4,601,903, 4,599,231,4,599,230, and 4,596,792, all incorporated herein by reference. Suchmethods may be used to prepare a vaccine comprising an antigeniccomposition comprising folate binding protein epitopes and/or variantsas active ingredient(s), in light of the present disclosure. Inpreferred embodiments, the compositions of the present invention areprepared to be pharmacologically acceptable vaccines.

Pharmaceutical vaccine compositions of the present invention comprise aneffective amount of one or more folate binding protein epitopes and/orvariants or additional agent dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of an pharmaceutical composition thatcontains at least one folate binding protein epitope or additionalactive ingredient will be known to those of skill in the art in light ofthe present disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. Moreover, for animal (e.g, human) administration, it will beunderstood that preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g, antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, binders, excipients, disintegration agents, lubricants,sweetening agents, flavoring agents, dyes, such like materials andcombinations thereof, as would be known to one of ordinary skill in theart (see, for example, Remington's Pharmaceutical Sciences, 18th Ed.Mack Printing Company, 1990, pp. 1289-1329, incorporated herein byreference). The folate binding protein variant may comprise differenttypes of carriers depending on whether it is to be administered insolid, liquid or aerosol form, and whether it need to be sterile forsuch routes of administration as injection. Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the therapeutic or pharmaceutical compositions is contemplated.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g, methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The folate binding protein variant may be formulated into a compositionin a free base, neutral or salt form. Pharmaceutically acceptable salts,include the acid addition salts, e.g, those formed with the free aminogroups of a proteinaceous composition, or which are formed withinorganic acids such as for example, hydrochloric or phosphoric acids,or such organic acids as acetic, oxalic, tartaric or mandelic acid.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as for example, sodium, potassium, ammonium,calcium or ferric hydroxides; or such organic bases as isopropylamine,trimethylamine, histidine or procaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g, glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g, triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

In other embodiments, one may use nasal solutions or sprays, aerosols orinhalants in the present invention. Such compositions are generallydesigned to be compatible with the target tissue type. In a non-limitingexample, nasal solutions are usually aqueous solutions designed to beadministered to the nasal passages in drops or sprays. Nasal solutionsare prepared so that they are similar in many respects to nasalsecretions, so that normal ciliary action is maintained. Thus, inpreferred embodiments the aqueous nasal solutions usually are isotonicor slightly buffered to maintain a pH of about 5.5 to about 6.5. Inaddition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments the folate binding protein variant is preparedfor administration by such routes as oral ingestion. In theseembodiments, the solid composition may comprise, for example, solutions,suspensions, emulsions, tablets, pills, capsules (e.g, hard or softshelled gelatin capsules), sustained release formulations, buccalcompositions, troches, elixirs, suspensions, syrups, wafers, orcombinations thereof. Oral compositions may be incorporated directlywith the food of the diet. Preferred carriers for oral administrationcomprise inert diluents, assimilable edible carriers or combinationsthereof. In other aspects of the invention, the oral composition may beprepared as a syrup or elixir. A syrup or elixir, and may comprise, forexample, at least one active agent, a sweetening agent, a preservative,a flavoring agent, a dye, a preservative, or combinations thereof.

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

J. Vaccine Administration

The manner of administration of a vaccine may be varied widely. Any ofthe conventional methods for administration of a vaccine are applicable.For example, a vaccine may be conventionally administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intratumorally, intramuscularly, intraperitoneally, subcutaneously,intravesicularlly, mucosally, intrapericardially, orally, rectally,nasally, topically, in eye drops, locally, using aerosol, injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in cremes, in lipid compositions(e.g, liposomes), or by other method or any combination of the forgoingas would be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference).

A vaccination schedule and dosages may be varied on a patient by patientbasis, taking into account, for example, factors such as the weight andage of the patient, the type of disease being treated, the severity ofthe disease condition, previous or concurrent therapeutic interventions,the manner of administration and the like, which can be readilydetermined by one of ordinary skill in the art.

A vaccine is administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. For example, the intramuscular route may be preferred inthe case of toxins with short half lives in vivo. The quantity to beadministered depends on the subject to be treated, including, e.g, thecapacity of the individual's immune system to synthesize antibodies, andthe degree of protection desired. The dosage of the vaccine will dependon the route of administration and will vary according to the size ofthe host. Precise amounts of an active ingredient required to beadministered depend on the judgment of the practitioner. In certainembodiments, pharmaceutical compositions may comprise, for example, atleast about 0.1% of an active compound. In other embodiments, the anactive compound may comprise between about 2% to about 75% of the weightof the unit, or between about 25% to about 60%, for example, and anyrange derivable therein However, a suitable dosage range may be, forexample, of the order of several hundred micrograms active ingredientper vaccination. In other non-limiting examples, a dose may alsocomprise from about 1 microgram/kg/body weight, about 5microgram/kg/body weight, about 10 microgram/kg/body weight, about 50microgram/kg/body weight, about 100 microgram/kg/body weight, about 200microgram/kg/body weight, about 350 microgram/kg/body weight, about 500microgram/kg/body weight, about 1 milligram/kg/body weight, about 5milligram/kg/body weight, about 10 milligram/kg/body weight, about 50milligram/kg/body weight, about 100 milligram/kg/body weight, about 200milligram/kg/body weight, about 350 milligram/kg/body weight, about 500milligram/kg/body weight, to about 1000 mg/kg/body weight or more pervaccination, and any range derivable therein. In non-limiting examplesof a derivable range from the numbers listed herein, a range of about 5mg/kg/body weight to about 100 mg/kg/body weight, about 5microgram/kg/body weight to about 500 milligram/kg/body weight, etc.,can be administered, based on the numbers described above. A suitableregime for initial administration and booster administrations (e.g,innoculations) are also variable, but are typified by an initialadministration followed by subsequent inoculation(s) or otheradministration(s).

In many instances, it will be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and preferably one or more, usually at leastabout three vaccinations. The vaccinations will normally be at from twoto twelve week intervals, more usually from three to five weekintervals. Periodic boosters at intervals of 1-5 years, usually threeyears, will be desirable to maintain protective levels of theantibodies.

The course of the immunization may be followed by assays for antibodiesfor the supernatant antigens. The assays may be performed by labelingwith conventional labels, such as radionuclides, enzymes, fluorescents,and the like. These techniques are well known and may be found in a widevariety of patents, such as U.S. Pat. Nos. 3,791,932; 4,174,384 and3,949,064, as illustrative of these types of assays. Other immune assayscan be performed and assays of protection from challenge with the folatebinding protein variant can be performed, following immunization.

K. Enhancement of an Immune Response

The present invention includes a method of enhancing the immune responsein a subject comprising the steps of contacting one or more lymphocyteswith a folate binding protein variant antigenic composition, wherein theantigen comprises as part of its sequence a sequence in accordance withSEQ ID NO:1 through SEQ ID NO:8, or a immunologically functionalequivalent thereof. In certain embodiments the one or more lymphocytesis comprised in an animal, such as a human. In other embodiments, thelymphocyte(s) may be isolated from an animal or from a tissue (e.g,blood) of the animal. In certain preferred embodiments, thelymphocyte(s) are peripheral blood lymphocyte(s). In certainembodiments, the one or more lymphocytes comprise a T-lymphocyte or aB-lymphocyte. In a particularly preferred facet, the T-lymphocyte is acytotoxic T-lymphocyte.

The enhanced immune response may be an active or a passive immuneresponse. Alternatively, the response may be part of an adoptiveimmunotherapy approach in which lymphocyte(s) are obtained with from ananimal (e.g, a patient), then pulsed with composition comprising anantigenic composition. In a preferred embodiment, the lymphocyte(s) maybe administered to the same or different animal (e.g, same or differentdonors).

1. Cytotoxic T Lymphocytes

In certain embodiments, T-lymphocytes are specifically activated bycontact with an antigenic composition of the present invention. Incertain embodiments, T-lymphocytes are activated by contact with anantigen presenting cell that is or has been in contact with an antigeniccomposition of the invention.

T cells express a unique antigen binding receptor on their membrane(T-cell receptor), which can only recognize antigen in association withmajor histocompatibility complex (MHC) molecules on the surface of othercells. There are several populations of T cells, such as T helper cellsand T cytotoxic cells. T helper cells and T cytotoxic cells areprimarily distinguished by their display of the membrane boundglycoproteins CD4 and CD8, respectively. T helper cells secret variouslymphokines, that are crucial for the activation of B cells, T cytotoxiccells, macrophages and other cells of the immune system. In contrast, aT cytotoxic cell that recognizes an antigen-MHC complex proliferates anddifferentiates into an effector cell called a cytotoxic T lymphocyte(CTL). CTLs eliminate cells of the body displaying antigen by producingsubstances that result in cell lysis.

CTL activity can be assessed by methods described herein or as would beknown to one of skill in the art. For example, CTLs may be assessed infreshly isolated peripheral blood mononuclear cells (PBMC), in aphytohaemaglutinin-stimulated IL-2 expanded cell line established fromPBMC (Bernard et al., 1998) or by T cells isolated from a previouslyimmunized subject and restimulated for 6 days with DC infected with anadenovirus vector containing antigen using standard 4 h 51^(Cr) releasemicrotoxicity assays. In another fluorometric assay developed fordetecting cell-mediated cytotoxicity, the fluorophore used is thenon-toxic molecule ALAMARBLUE (dye) (Nociari et al., 1998). ThealamarBlue ALAMARBLUE (dye) is fluorescently quenched (i.e., low quantumyield) until mitochondrial reduction occurs, which then results in adramatic increase in the alamarBlue fluorescence intensity (i.e.,increase in the quantum yield). This assay is reported to be extremelysensitive, specific and requires a significantly lower number ofeffector cells than the standard 51^(Cr) release assay.

In certain aspects, T helper cell responses can be measured by in vitroor in vivo assay with peptides, polypeptides or proteins. In vitroassays include measurement of a specific cytokine release by enzyme,radioisotope, chromaphore or fluorescent assays. In vivo assays includedelayed type hypersensitivity responses called skin tests, as would beknown to one of ordinary skill in the art.

2. Antigen Presenting Cells

In general, the term “antigen presenting cell” can be any cell thataccomplishes the goal of the invention by aiding the enhancement of animmune response (i.e., from the T-cell or -B-cell arms of the immunesystem) against an antigen (e.g, a folate binding protein variant or aimmunologically functional equivalent) or antigenic composition of thepresent invention. Such cells can be defined by those of skill in theart, using methods disclosed herein and in the art. As is understood byone of ordinary skill in the art (see for example Kuby, 1993,incorporated herein by reference), and used herein certain embodiments,a cell that displays or presents an antigen normally or preferentiallywith a class II major histocompatability molecule or complex to animmune cell is an “antigen presenting cell.” In certain aspects, a cell(e.g, an APC cell) may be fused with another cell, such as a recombinantcell or a tumor cell that expresses the desired antigen. Methods forpreparing a fusion of two or more cells is well known in the art, suchas for example, the methods disclosed in Goding, pp. 65-66, 71-74 1986;Campbell, pp. 75-83, 1984; Kohler and Milstein, 1975; Kohler andMilstein, 1976, Gefter et al., 1977, each incorporated herein byreference. In some cases, the immune cell to which an antigen presentingcell displays or presents an antigen to is a CD4⁺ TH cell. Additionalmolecules expressed on the APC or other immune cells may aid or improvethe enhancement of an immune response. Secreted or soluble molecules,such as for example, immunomodulators and adjuvants, may also aid orenhance the immune response against an antigen. Such molecules are wellknown to one of skill in the art, and various examples are describedherein.

VII. Peptide Formulations

Peptides containing the epitope motifs described herein are contemplatedfor use in therapeutics to provide universal FBP targets and antigensfor CTLs in the HLA-A2 system. The development of therapeutics based onthese novel sequences provides induction of tumor reactive immune cellsin vivo through the formulation of synthetic cancer vaccines, as well asinduction of tumor-reactive T-cells in vitro through eitherpeptide-mediated (e.g, lipopeptide) or cell-mediated (e.g, EBV-B linesusing either autologous or HLA-A2 transfectants where the gene for thepeptide of interest is introduced, and the peptide is expressedassociated with HLA-A2 on the surface). The use of these novel peptidesas components of vaccines to prevent, or lessen the chance of cancerprogression is also contemplated.

The peptides contemplated for use, being smaller than othercompositions, such as envelope proteins, will have improvedbioavailability and half lives. If desired, stability examinations maybe performed on the peptides, including, e.g, pre-incubation in humanserum and plasma; treatment with various proteases; and alsotemperature- and pH-stability analyses. If found to be necessary, thestability of the synthetic peptides may be enhanced by any one of avariety of methods such as, for example, employing D-amino acids inplace of L-amino acids for peptide synthesis; using blocking groups liket-boc and the like; or encapsulating the peptides within liposomes. Thebio-availability of select mixtures of peptides may also be determinedby injecting radio-labeled peptides into experimental animals, such asmice and/or Rhesus monkeys, and subsequently analyzing their tissuedistribution.

If stability enhancement was desired, it is contemplated that the use ofdextrorotary amino acids (D-amino acids) would be advantageous as thiswould result in even longer bioavailability due to the inability ofproteases to attack these types of structures. The peptides of thepresent invention may also be further stabilized, for example, by theaddition of groups to the N- or C-termini, such as by acylation oramination. If desired, the peptides could even be in the form oflipid-tailed peptides, formulated into surfactant-like micelles, orother peptide multimers. The preparation of peptide multimers andsurfactant-like micelles is described in detail in U.S. Ser. No.07/945,865, incorporated herein by reference. The compositions of thepresent invention are contemplated to be particularly advantageous foruse in economical and safe anti-tumor/anti-cancer therapeutics, andspecific therapeutic formulations may be tested in experimental animalmodels, such as mice, rats, rabbits, guinea pigs, cats, goats, Rhesusmonkeys, chimpanzees, and the like, in order to determine more preciselythe dosage forms required.

In addition to the peptidyl compounds described herein, the inventorsalso contemplate that other sterically similar compounds may beformulated to mimic the key portions of the peptide structure and thatsuch compounds may also be used in the same manner as the peptides ofthe invention. This may be achieved by the techniques of modelling andchemical design known to those of skill in the art. For example,esterification and other alkylations may be employed to modify theterminus of a peptide to mimic a particular terminal motif structure. Itwill be understood that all such sterically similar constructs fallwithin the scope of the present invention.

Therapeutic or pharmacological compositions of the present inventionwill generally comprise an effective amount of a CTL-stimulating peptideor peptides, dissolved or dispersed in a pharmaceutically acceptablemedium. The phrase “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce an allergic, toxic, orotherwise adverse reaction when administered to a human.Pharmaceutically acceptable media or carriers include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated.

Supplementary active ingredients can also be incorporated into thetherapeutic compositions of the present invention. For example, thestimulatory peptides may also be combined with peptides includingcytotoxic T-cell- or T-helper-cell-inducing epitopes (as disclosed inU.S. Ser. No. 07/945,865; incorporated herein by reference) to createpeptide cocktails for immunization and treatment.

The preparation of pharmaceutical or pharmacological compositionscontaining a CTL-stimulating peptide or peptides, includingdextrorotatory peptides, as active ingredients will be known to those ofskill in the art in light of the present disclosure. Typically, suchcompositions may be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection; as tablets or other solids for oraladministration; as time release capsules; or in any other form currentlyused, including cremes, lotions, mouthwashes, inhalents and the like.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

Sterile solutions suitable for intravenous administration are preferredin certain embodiments and are contemplated to be particularly effectivein stimulating CTLs and/or producing an immune response in an animal.The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

A peptide or peptides can be formulated into a composition in a neutralor salt form. Pharmaceutically acceptable salts, include the acidaddition salts (formed with the free amino groups of the peptide) andwhich are formed with inorganic acids such as, e.g, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine, and thelike.

The carrier can also be a solvent or dispersion medium containing, e.g,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained by inter aliathe use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought inter alia by various antibacterial ad antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, e.g, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The preparation of more- or highly-concentrated solutions forintramuscular injection is also contemplated. This is envisioned to haveparticular utility in facilitating the treatment of needle stickinjuries to animals or even humans. In this regard, the use of DMSO assolvent is preferred as this will result in extremely rapid penetration,delivering high concentrations of the active peptide, peptides or agentsto a small area.

The use of sterile formulations, such as saline-based washes, byveterinarians, technicians, surgeons, physicians or health care workersto cleanse a particular area in the operating field may also beparticularly useful. Therapeutic formulations in accordance with thepresent invention may also be reconstituted in the form of mouthwashes,including the peptides alone, or in conjunction with antifungalreagents. Inhalant forms are also envisioned, which again, may containactive peptides or agents alone, or in conjunction with other agents,such as, e.g, pentamidine. The therapeutic formulations of the inventionmay also be prepared in forms suitable for topical administration, suchas in cremes and lotions.

Suitable preservatives for use in such a solution include benzalkoniumchloride, benzethonium chloride, chlorobutanol, thimerosal and the like.Suitable buffers include boric acid, sodium and potassium bicarbonate,sodium and potassium borates, sodium and potassium carbonate, sodiumacetate, sodium biphosphate and the like, in amounts sufficient tomaintain the pH at between about pH 6 and pH 8, and preferably, betweenabout pH 7 and pH 7.5. Suitable tonicity agents are dextran 40, dextran70, dextrose, glycerin, potassium chloride, propylene glycol, sodiumchloride, and the like, such that the sodium chloride equivalent of theophthalmic solution is in the range 0.9±0.2%. Suitable antioxidants andstabilizers include sodium bisulfite, sodium metabisulfite, sodiumthiosulfate, thiourea and the like. Suitable wetting and clarifyingagents include polysorbate 80, polysorbate 20, poloxamer 282 andtyloxapol. Suitable viscosity-increasing agents include dextran 40,dextran 70, gelatin, glycerin, hydroxyethylcellulose,hydroxmethyl-propylcellulose, lanolin, methylcellulose, petrolatum,polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone,carboxymethylcellulose and the like.

Upon formulation, therapeutics will be administered in a mannercompatible with the dosage formulation, and in such amount as ispharmacologically effective. The formulations are easily administered ina variety of dosage forms, such as the type of injectable solutionsdescribed above, but drug release capsules and the like can also beemployed. As used herein, “pharmacologically effective amount” means anamount of composition is used that contains an amount of a peptide orpeptides sufficient to significantly stimulate a CTL or generate animmune response in an animal.

In this context, the quantity of peptide(s) and volume of composition tobe administered depends on the host animal to be treated, such as, thecapacity of the host animal's immune system to produce an immuneresponse. Precise amounts of active peptide required to be administereddepend on the judgment of the practitioner and are peculiar to eachindividual.

A minimal volume of a composition required to disperse the peptide istypically utilized. Suitable regimes for administration are alsovariable, but would be typified by initially administering the compoundand monitoring the results and then giving further controlled doses atfurther intervals. For example, for parenteral administration, asuitably buffered, and if necessary, isotonic aqueous solution would beprepared and used for intravenous, intramuscular, subcutaneous or evenintraperitoneal administration. One dosage could be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580).

In certain embodiments, active compounds may be administered orally.This is contemplated for agents that are generally resistant, or havebeen rendered resistant, to proteolysis by digestive enzymes. Suchcompounds are contemplated to include chemically designed or modifiedagents; dextrorotatory peptides; and peptide and liposomal formulationsin timed-release capsules to avoid peptidase, protease and/or lipasedegradation.

Oral formulations may include compounds in combination with an inertdiluent or an edible carrier which may be assimilated; those enclosed inhard- or soft-shell gelatin capsules; those compressed into tablets; orthose incorporated directly with the food of the diet. For oraltherapeutic administration, the active compounds may be incorporatedwith excipients and used in the form of ingestible tablets, buccaltables, troches, capsules, elixirs, suspensions, syrups, wafers, and thelike. Such compositions and preparations should generally contain atleast 0.1% of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 60% of the weight of the unit. The amount of activecompounds in such therapeutically useful compositions is such that asuitable dosage will be obtained.

Tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, corn starch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent, such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparaben as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

The peptides may be used in their immunizing capacity by administeringan amount effective to generate an immune response in an animal. In thissense, such an “amount effective to generate an immune response” meansan amount of composition that contains a peptide or peptide mixturesufficient to significantly produce an antigenic response in the animal.

VIII. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Rationale for Variant Design

Studies in experimental models regarding lymphocyte development in thethymus show that interaction of thymocytes with weak or null (noapparent effect) agonists lead to positive selection (i.e. survival) ofresponders for a specific Ag, while stimulation with strong agonistsleads to negative selection (deletion of reactive CTL). Similarly,recent studies on CD8⁺ cell responses from peripheral blood show that Agvariants with null or weak agonistic activity induced expansion ofprecursors of CTL responding to a model Ag, but not effector function.These results were obtained with transgenic animals, and the recipientsfor the CTL were heavily irradiated. There is little informationconcerning how the responders to tumor, and/or their precursors, can bemaintained and avoid elimination in healthy individuals, or patientswithout evidence of disease. However, the presence of such precursors,or of activated CTL recognizing tumor Ag, (Peoples et al., 1998; Hudsonet al., 1998; Peoples et al, 1998; Kim et al., 1999; Lee et al., 2000)is proof that such responders exist in the peripheral blood. Approachesto promote their survival, expansion and induction of lytic formation isbeneficial for the patients. If the responders targeted for survival arelow-affinity CTL, the weak affinity is expected to be compensated by asignificant increase in effector numbers. If the responders are of highaffinity, protection from AICD will also allow their expansion.

To design “survival inducing” Ag, the present invention focuses on theFBP epitope E39: EIWTHSYKV (SEQ ID NO: 268). This epitope is recognized,although with low affinity, by ovarian and breast tumor reactive CTL. Itwas predicted that improved immunogenicity in terms of net gain in cellnumbers reacting with the wild-type Ag is achieved by reducing thepositive charge at the amino acid in position 5 (histidine) andreplacement of histidine with phenylalanine (Phe). Phe is not charged,but its benzene aromatic ring is a close substitution for the imidazolering of histidine. To ensure a better flexibility of the residues in thepeptide, the phenolic structure of tyrosine was replaced with thealiphatic core chain of Threonine (Thr). Both Tyr and Thr contain an OH(hydroxyl) side chain group. Thus, the positive charge in position 5 andthe rigid structure of Tyr were eliminated. In a specific embodiment,this increases the flexibility of the residues 5-9 (SYKV) (SEQ ID NO:270) in the peptide and allows for a better fitting of the TCR with thepeptide MHC complex. The variant: E I W T F S T KV (SEQ ID NO: 5) wasdesignated J65. Additional variants of J65 were created with changes inposition 7 (Tyr)→*Thr only=designated J77, in position 5 onlyPhe→*His=designated J78, and in positions 1 and 6. Theseanalogs/variants are listed in Table 5.

TABLE 5 Variants of Folate Binding Protein VARIANT SEQUENCE CHANGE E39EIWTHSYKV (SEQ wild type ID NO: 268) J77 EIWTHSTKV (SEQ Y7→T ID NO: 1)J78 EIWTFSYKV (SEQ ID H5→F NO: 2) J68 FIWTFATKV (SEQ IDE1→F, H5→F, S6→A; NO: 3) Y7→T J67 EIWTHATKV (SEQ S6→A, Y7→T ID NO: 4)J66 FIWTFSTKV (SEQ ID E1→F, H5→F, Y7→T NO: 271) J65 EIWTFSTKV (SEQ IDH5→F, Y7→T NO: 5) J64 GIWTHSTKV (SEQ E1→G, Y7→T ID NO: 7) J63FIWTHSTKV (SEQ ID E1→F, Y7→T NO: 8)

Selection of these Ag variants was made on the principle of Agalteration aiming to alternate signaling. In addition to substitutionsH→F (Pos. 5) and Y→T (pos. 7), substitutions were introduced in theother positions: S→A (Pos. 6 and Glu (B)→F and E→Gly (G) (in Pos. 1).The purpose of these substitutions was to remove potential reactinggroups with the TCR. In the substitution S→A (Pos. A), this changeremoves a side chain OH group. In position 1, the substitution E(glutamic acid)→glycine, removes the entire aliphatic side chain plusthe charged COO group. Also in position 1, the substitution E→F (removesthe charged group COO, but introduces an aromatic ring). Thesesubstitutions aim to diminish the reactivity of the peptide with theTCR.

Example 2

IFN-γ Induction and CTL Activity

The HLA-A2 stabilizing ability of the variant peptides has also beendetermined (FIG. 1). The results show that the stabilizing ability ofJ65 is almost half of the stabilizing ability of E39. In contrast,substitutions at position 1 increase the binding affinity of thepeptide. The results in FIG. 2 show the cytolytic activity ofJ65-induced CTL compared with E39-induced CTL. The results indicate thatJ65 was a weaker inducer of IFN-γ from 3×J65 stimulated cultures thanJ77 and E39, suggesting that the changes in the sequence had cumulativeeffects in decreasing IFN-γ induction.

To address the effects of FBP variants on induction of CTL activity,PBMC cultures from the healthy donor stimulated three times with J65were split in three and restimulated with either E39 or J65 or J77. Acontrol culture was made of the same PBMC stimulated three times withE39 and restimulated with E39 for the fourth time. PBMC stimulated threetimes with E39 (3×E39) followed by E39 showed moderate weak recognitionof E39. In contrast, 3×J65 stimulated CTL showed significantly higherrecognition of E39 after stimulation with E39. A similar picture wasobserved with 3×J65 cells restimulated with J65, while 3×J65restimulated with J77 showed significantly lower CTL activity than 3×J65stimulated with the other peptides. It was recently reported that memoryCTL reacting with the tumor Ag such as FBP are present in the blood ofhealthy individuals (Lee et al., 2000). These cells can be easilyactivated by stimulation with the corresponding peptide presented ondendritic cells (Kim et al., 1999). To evaluate the stimulating abilityof the analogs J65 and J77, PBMC from a responding donor were stimulatedwith E39, J65 and J77. These results show that the potentiating role ofJ65 in responder proliferation and cytotoxicity does not reflectenhanced IL-2 and/or IFN-γ secretion compared with the wild-type Ag, butits weaker cytokine-inducing activity appears to protect CTL of higheraffinity from apoptosis by avoiding overstimulation.

Example 3 Specific IL-2 Induction by Priming with FBP Variants

In J65-primed CTL, higher CTL activity and IFN-γ secretion can beelicited by the wild-type epitope E39, suggesting a protective effect ofthe previous stimulations. The results in FIG. 3 show that J65 and J77induced lower levels of IL-2 in the PBMC of this donor compared with thewild-type peptide E39. To identify which of E39 variants induced highercell expansion, PBMC from the same donor were stimulated three timeswith the corresponding peptide, and the resulting live cells werecounted a week after each stimulation. The results in FIG. 4 show thatcultures stimulated with E39 initially expanded faster than othercultures; however, after the third stimulation, cultures stimulated withJ65 increased faster in numbers. In contrast, cultures stimulated withJ78 (H→F) and J77 (Y→T) proliferated slower than control cultures whichwere not stimulated with peptide. Similar results were obtained with J65in another donor (FIG. 5). In this donor, cells stimulated with E39 diedafter the third stimulation while cells stimulated by J65 expandedfaster. Cells stimulated with J77 and J78 also expanded, but at a slowerrate.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

Patents

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All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as describedherein.

The invention claimed is:
 1. A method of inducing immunity against acancer expressing folate binding protein (SEQ ID NO:10) in anindividual, the method comprising administering to the individual aneffective amount of a first peptide comprising the amino acid sequenceof a variant of a peptide fragment of the polypeptide of SEQ ID NO: 10,wherein the peptide is of at least 9 amino acids or up to about 30 aminoacids and is selected from the group consisting of: a) a peptidecomprising the amino acid sequence of SEQ ID NO: 268 but for thesubstitution therein of amino acids at positions 5 and 7 thereof byphenylalanine and threonine, respectively, and from 0 to 21 additionalcontiguous amino acids of SEQ ID NO: 10 that flank SEQ ID NO: 268 ascontained therein; b) a peptide comprising the amino acid sequence ofSEQ ID NO:268 but for substitution therein of the amino acid at position5 thereof by phenylalanine, and from 0 to 21 additional contiguous aminoacids of SEQ ID NO: 10 that flank SEQ ID NO: 268 as contained therein;c) a peptide comprising the amino acid sequence of SEQ ID NO:268 but forsubstitution therein of the amino acids at positions 1, 5, 6, and 7thereof by phenylalanine, phenylalanine, alanine, and threonine,respectively, and from 0 to 21 additional contiguous amino acids of SEQID NO: 10 that flank SEQ ID NO: 268 as contained therein; d) a peptidecomprising the amino acid sequence of SEQ ID NO:268 but for substitutiontherein of the amino acids at positions 6 and 7 thereof by alanine andthreonine, respectively, and from 0 to 21 additional contiguous aminoacids of SEQ ID NO: 10 that flank SEQ ID NO: 268 as contained therein;e) a peptide comprising the amino acid sequence of SEQ ID NO:268 but forsubstitution therein of the amino acids at positions 1 and 7 thereof byphenylalanine and threonine, respectively, and from 0 to 21 additionalcontiguous amino acids of SEQ ID NO: 10 that flank SEQ ID NO: 268 ascontained therein; f) a peptide comprising the amino acid sequence ofSEQ ID NO:268 but for substitution therein of the amino acids atpositions 1, 5, and 7 thereof by phenylalanine, phenylalanine, andthreonine, respectively, and from 0 to 21 additional contiguous aminoacids of SEQ ID NO: 10 that flank SEQ ID NO: 268 as contained therein;and g) a peptide comprising the amino acid sequence of SEQ ID NO:268 butfor substitution therein of the amino acids at positions 1 and 7 thereofby glycine and threonine, respectively, and from 0 to 21 additionalcontiguous amino acids of SEQ ID NO: 10 that flank SEQ ID NO: 268 ascontained therein.
 2. The method of claim 1, further comprisingadministering a second peptide of at least 9 amino acids or up to about30 amino acids selected from the group consisting of: a) a peptidecomprising the amino acid sequence of SEQ ID NO: 268 but for thesubstitution therein of amino acids at positions 5 and 7 thereof byphenylalanine and threonine, respectively, and from 0 to 21 additionalcontiguous amino acids of SEQ ID NO: 10 that flank SEQ ID NO: 268 ascontained therein; b) a peptide comprising the amino acid sequence ofSEQ ID NO:268 but for substitution therein of the amino acid at position5 thereof by phenylalanine, and from 0 to 21 additional contiguous aminoacids of SEQ ID NO: 10 that flank SEQ ID NO: 268 as contained therein;c) a peptide comprising the amino acid sequence of SEQ ID NO:268 but forsubstitution therein of the amino acids at positions 1, 5, 6, and 7thereof by phenylalanine, phenylalanine, alanine, and threonine,respectively, and from 0 to 21 additional contiguous amino acids of SEQID NO: 10 that flank SEQ ID NO: 268 as contained therein; d) a peptidecomprising the amino acid sequence of SEQ ID NO:268 but for substitutiontherein of the amino acids at positions 6 and 7 thereof by alanine andthreonine, respectively, and from 0 to 21 additional contiguous aminoacids of SEQ ID NO: 10 that flank SEQ ID NO: 268 as contained therein;e) a peptide comprising the amino acid sequence of SEQ ID NO:268 but forsubstitution therein of the amino acids at positions 1 and 7 thereof byphenylalanine and threonine, respectively, and from 0 to 21 additionalcontiguous amino acids of SEQ ID NO: 10 that flank SEQ ID NO: 268 ascontained therein; f) a peptide comprising the amino acid sequence ofSEQ ID NO:268 but for substitution therein of the amino acids atpositions 1, 5, and 7 thereof by phenylalanine, phenylalanine, andthreonine, respectively, and from 0 to 21 additional contiguous aminoacids of SEQ ID NO: 10 that flank SEQ ID NO: 268 as contained therein;g) a peptide comprising the amino acid sequence of SEQ ID NO:268 but forsubstitution therein of the amino acids at positions 1 and 7 thereof byglycine and threonine, respectively, and from 0 to 21 additionalcontiguous amino acids of SEQ ID NO: 10 that flank SEQ ID NO: 268 ascontained therein; h) a peptide comprising the amino acid sequence ofSEQ ID NO:268 but for substitution therein of the amino acid at position7 thereof by threonine, and from 0 to 21 additional contiguous aminoacids of SEQ ID NO: 10 that flank SEQ ID NO: 268 as contained therein;i) a peptide comprising the amino acid sequence of SEQ ID NO:268 andfrom 0 to 21 additional contiguous amino acids of SEQ ID NO: 10 thatflank SEQ ID NO: 268 as contained therein; and j) a peptide comprisingthe amino acid sequence of SEQ ID NO:269 and from 0 to 21 additionalcontiguous amino acids of SEQ ID NO: 10 that flank SEQ ID NO: 269 ascontained therein.
 3. The method of claim 2, wherein the first peptideis administered prior to the administration of the second peptide. 4.The method of claim 2, wherein the first peptide is administeredsubsequent to the administration of the second peptide.
 5. The method ofclaim 2, wherein the first peptide is administered both prior to andsubsequent to the administration of the second peptide.
 6. The method ofclaim 2, wherein the second peptide consists of an amino acid sequenceselected from the group consisting of SEQ ID NO:268 (E39), SEQ ID NO:269(E41), SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:271.
 7. The method of claim 1,wherein the individual is substantially susceptible to recurrence ofcancer.
 8. The method of claim 1, wherein the cancer is breast cancer,ovarian cancer, endometrial cancer, colorectal cancer, lung cancer,renal cancer, melanoma, kidney cancer, prostate cancer, brain cancer,sarcomas, or a combination thereof.
 9. The method of claim 1, furthercomprising administering an adjuvant.
 10. The method of claim 1, furthercomprising administering a booster comprising one or more peptidesselected from the group consisting of a) a peptide comprising the aminoacid sequence of SEQ ID NO: 268 but for the substitution therein ofamino acids at positions 5 and 7 thereof by phenylalanine and threonine,respectively, and from 0 to 21 additional contiguous amino acids of SEQID NO: 10 that flank SEQ ID NO: 268 as contained therein; b) a peptidecomprising the amino acid sequence of SEQ ID NO:268 but for substitutiontherein of the amino acid at position 5 thereof by phenylalanine, andfrom 0 to 21 additional contiguous amino acids of SEQ ID NO: 10 thatflank SEQ ID NO: 268 as contained therein; c) a peptide comprising theamino acid sequence of SEQ ID NO:268 but for substitution therein of theamino acids at positions 1, 5, 6, and 7 thereof by phenylalanine,phenylalanine, alanine, and threonine, respectively, and from 0 to 21additional contiguous amino acids of SEQ ID NO: 10 that flank SEQ ID NO:268 as contained therein; d) a peptide comprising the amino acidsequence of SEQ ID NO:268 but for substitution therein of the aminoacids at positions 6 and 7 thereof by alanine and threonine,respectively, and from 0 to 21 additional contiguous amino acids of SEQID NO: 10 that flank SEQ ID NO: 268 as contained therein; e) a peptidecomprising the amino acid sequence of SEQ ID NO:268 but for substitutiontherein of the amino acids at positions 1 and 7 thereof by phenylalanineand threonine, respectively, and from 0 to 21 additional contiguousamino acids of SEQ ID NO: 10 that flank SEQ ID NO: 268 as containedtherein; f) a peptide comprising the amino acid sequence of SEQ IDNO:268 but for substitution therein of the amino acids at positions 1,5, and 7 thereof by phenylalanine, phenylalanine, and threonine,respectively, and from 0 to 21 additional contiguous amino acids of SEQID NO: 10 that flank SEQ ID NO: 268 as contained therein; g) a peptidecomprising the amino acid sequence of SEQ ID NO:268 but for substitutiontherein of the amino acids at positions 1 and 7 thereof by glycine andthreonine, respectively, and from 0 to 21 additional contiguous aminoacids of SEQ ID NO: 10 that flank SEQ ID NO: 268 as contained therein;h) a peptide comprising the amino acid sequence of SEQ ID NO:268 but forsubstitution therein of the amino acid at position 7 thereof bythreonine, and from 0 to 21 additional contiguous amino acids of SEQ IDNO: 10 that flank SEQ ID NO: 268 as contained therein; i) a peptidecomprising the amino acid sequence of SEQ ID NO:268 and from 0 to 21additional contiguous amino acids of SEQ ID NO: 10 that flank SEQ ID NO:268 as contained therein; and j) a peptide comprising the amino acidsequence of SEQ ID NO:269 and from 0 to 21 additional contiguous aminoacids of SEQ ID NO: 10 that flank SEQ ID NO: 269 as contained therein.11. The method of claim 1, wherein the peptide is formulated foradministration parenterally, topically, or as an inhalant, aerosol orspray.
 12. A method for stimulating cytotoxic T-lymphocytes to folatebinding protein (SEQ ID NO:10), comprising the step of contacting thecytotoxic T-lymphocytes with an effective amount of a peptide comprisingthe amino acid sequence of a variant of a peptide fragment of thepolypeptide of SEQ ID NO: 10, wherein the peptide is of at least 9 aminoacids or up to about 30 amino acids and is selected from the groupconsisting of: a) a peptide comprising the amino acid sequence of SEQ IDNO: 268 but for the substitution therein of amino acids at positions 5and 7 thereof by phenylalanine and threonine, respectively, and from 0to 21 additional contiguous amino acids of SEQ ID NO: 10 that flank SEQID NO: 268 as contained therein; b) a peptide comprising the amino acidsequence of SEQ ID NO:268 but for substitution therein of the amino acidat position 5 thereof by phenylalanine, and from 0 to 21 additionalcontiguous amino acids of SEQ ID NO: 10 that flank SEQ ID NO: 268 ascontained therein; c) a peptide comprising the amino acid sequence ofSEQ ID NO:268 but for substitution therein of the amino acids atpositions 1, 5, 6, and 7 thereof by phenylalanine, phenylalanine,alanine, and threonine, respectively, and from 0 to 21 additionalcontiguous amino acids of SEQ ID NO: 10 that flank SEQ ID NO: 268 ascontained therein; d) a peptide comprising the amino acid sequence ofSEQ ID NO:268 but for substitution therein of the amino acids atpositions 6 and 7 thereof by alanine and threonine, respectively, andfrom 0 to 21 additional contiguous amino acids of SEQ ID NO: 10 thatflank SEQ ID NO: 268 as contained therein; e) a peptide comprising theamino acid sequence of SEQ ID NO:268 but for substitution therein of theamino acids at positions 1 and 7 thereof by phenylalanine and threonine,respectively, and from 0 to 21 additional contiguous amino acids of SEQID NO: 10 that flank SEQ ID NO: 268 as contained therein; f) a peptidecomprising the amino acid sequence of SEQ ID NO:268 but for substitutiontherein of the amino acids at positions 1, 5, and 7 thereof byphenylalanine, phenylalanine, and threonine, respectively, and from 0 to21 additional contiguous amino acids of SEQ ID NO: 10 that flank SEQ IDNO: 268 as contained therein; and g) a peptide comprising the amino acidsequence of SEQ ID NO:268 but for substitution therein of the aminoacids at positions 1 and 7 thereof by glycine and threonine,respectively, and from 0 to 21 additional contiguous amino acids of SEQID NO: 10 that flank SEQ ID NO: 268 as contained therein.
 13. The methodof claim 12, wherein the cytotoxic T-lymphocytes are located within ahuman.
 14. The method of claim 12, further comprising administering apeptide fragment of the polypeptide of SEQ ID NO:10, wherein saidpeptide fragment is of at least 9 amino acids or up to about 30 aminoacids, and comprises SEQ ID NO:268.
 15. A method of generating an immuneresponse to folate binding protein (SEQ ID NO:10), comprising the stepof administering to a human a pharmaceutical composition comprising animmunologically effective amount of a peptide comprising the amino acidsequence of a variant of a peptide fragment of the polypeptide of SEQ IDNO: 10, wherein the peptide is of at least 9 amino acids or up to about30 amino acids and is selected from the group consisting of: a) apeptide comprising the amino acid sequence of SEQ ID NO: 268 but for thesubstitution therein of amino acids at positions 5 and 7 thereof byphenylalanine and threonine, respectively, and from 0 to 21 additionalcontiguous amino acids of SEQ ID NO: 10 that flank SEQ ID NO: 268 ascontained therein; b) a peptide comprising the amino acid sequence ofSEQ ID NO:268 but for substitution therein of the amino acid at position5 thereof by phenylalanine, and from 0 to 21 additional contiguous aminoacids of SEQ ID NO: 10 that flank SEQ ID NO: 268 as contained therein;c) a peptide comprising the amino acid sequence of SEQ ID NO:268 but forsubstitution therein of the amino acids at positions 1, 5, 6, and 7thereof by phenylalanine, phenylalanine, alanine, and threonine,respectively, and from 0 to 21 additional contiguous amino acids of SEQID NO: 10 that flank SEQ ID NO: 268 as contained therein; d) a peptidecomprising the amino acid sequence of SEQ ID NO:268 but for substitutiontherein of the amino acids at positions 6 and 7 thereof by alanine andthreonine, respectively, and from 0 to 21 additional contiguous aminoacids of SEQ ID NO: 10 that flank SEQ ID NO: 268 as contained therein;e) a peptide comprising the amino acid sequence of SEQ ID NO:268 but forsubstitution therein of the amino acids at positions 1 and 7 thereof byphenylalanine and threonine, respectively, and from 0 to 21 additionalcontiguous amino acids of SEQ ID NO: 10 that flank SEQ ID NO: 268 ascontained therein; f) a peptide comprising the amino acid sequence ofSEQ ID NO:268 but for substitution therein of the amino acids atpositions 1, 5, and 7 thereof by phenylalanine, phenylalanine, andthreonine, respectively, and from 0 to 21 additional contiguous aminoacids of SEQ ID NO: 10 that flank SEQ ID NO: 268 as contained therein;and g) a peptide comprising the amino acid sequence of SEQ ID NO:268 butfor substitution therein of the amino acids at positions 1 and 7 thereofby glycine and threonine, respectively, and from 0 to 21 additionalcontiguous amino acids of SEQ ID NO: 10 that flank SEQ ID NO: 268 ascontained therein.
 16. A method of inducing memory cytotoxicT-lymphocytes to folate binding protein (SEQ ID NO:10) in an individualcomprising the step of administering an effective amount of a peptidecomprising the amino acid sequence of a variant of a peptide fragment ofthe polypeptide of SEQ ID NO: 10, wherein the peptide is of at least 9amino acids or up to about 30 amino acids and is selected from the groupconsisting of: a) a peptide comprising the amino acid sequence of SEQ IDNO: 268 but for the substitution therein of amino acids at positions 5and 7 thereof by phenylalanine and threonine, respectively, and from 0to 21 additional contiguous amino acids of SEQ ID NO: 10 that flank SEQID NO: 268 as contained therein; b) a peptide comprising the amino acidsequence of SEQ ID NO:268 but for substitution therein of the amino acidat position 5 thereof by phenylalanine, and from 0 to 21 additionalcontiguous amino acids of SEQ ID NO: 10 that flank SEQ ID NO: 268 ascontained therein; c) a peptide comprising the amino acid sequence ofSEQ ID NO:268 but for substitution therein of the amino acids atpositions 1, 5, 6, and 7 thereof by phenylalanine, phenylalanine,alanine, and threonine, respectively, and from 0 to 21 additionalcontiguous amino acids of SEQ ID NO: 10 that flank SEQ ID NO: 268 ascontained therein; d) a peptide comprising the amino acid sequence ofSEQ ID NO:268 but for substitution therein of the amino acids atpositions 6 and 7 thereof by alanine and threonine, respectively, andfrom 0 to 21 additional contiguous amino acids of SEQ ID NO: 10 thatflank SEQ ID NO: 268 as contained therein; e) a peptide comprising theamino acid sequence of SEQ ID NO:268 but for substitution therein of theamino acids at positions 1 and 7 thereof by phenylalanine and threonine,respectively, and from 0 to 21 additional contiguous amino acids of SEQID NO: 10 that flank SEQ ID NO: 268 as contained therein; f) a peptidecomprising the amino acid sequence of SEQ ID NO:268 but for substitutiontherein of the amino acids at positions 1, 5, and 7 thereof byphenylalanine, phenylalanine, and threonine, respectively, and from 0 to21 additional contiguous amino acids of SEQ ID NO: 10 that flank SEQ IDNO: 268 as contained therein; and g) a peptide comprising the amino acidsequence of SEQ ID NO:268 but for substitution therein of the aminoacids at positions 1 and 7 thereof by glycine and threonine,respectively, and from 0 to 21 additional contiguous amino acids of SEQID NO: 10 that flank SEQ ID NO: 268 as contained therein.
 17. The methodof claim 1, wherein the first peptide consists of an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:271.